Phase-sensitively detected reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins

Abstract

The present invention discloses eleven reduced dimensionality (RD) triple resonance nuclear magnetic resonance (NMR) experiments for measuring chemical shift values of certain nuclei in a protein molecule, where the chemical shift values encoded in a peak pair of an NMR spectrum are detected in a phase sensitive manner. The RD 3D HA , CA ,(CO),N,HN NMR and RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiments are designed to yield “sequential” connectivities, while the RD 3D H α/β , C α/β , CO,HA NMR and RD 3D H α/β , C α/β ,N,HN NMR experiments provide “intraresidue” connectivities. The RD 3D H , C ,C,H-COSY NMR, RD 3D H , C ,C,H-TOCSY NMR, and RD 2D H , C ,H-COSY NMR experiments allow one to obtain assignments for aliphatic and aromatic side chain chemical shifts, while the RD 2D HB , CB ,(CG,CD),HD NMR experiment provide information for the aromatic side chain chemical shifts. In addition, methods of conducting suites of RD triple resonance NMR experiments for high-throughput resonance assignment of proteins and determination of secondary structure elements are disclosed.

1 . A method of conducting a reduced dimensionality three-dimensional (3D)  HA , CA ,(CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) an α-proton of amino acid residue i−1,  1 H α   i−1 ; (2) an α-carbon of amino acid residue i−1,  13 C α   i−1 ; (3) a polypeptide backbone amide nitrogen of amino acid residue i,  15 N i ; and (4) a polypeptide backbone amide proton of amino acid residue i,  1 H N   i , wherein the chemical shift values of  1 H α   i−1  and  13 C α   i−1  which are encoded in a peak pair of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H α   i−1  and  13 C α   i−1  of amino acid residue i−1 are connected to the chemical shift evolutions of  15 N i  and  1 H N   i  of amino acid residue i, under conditions effective (1) to generate NMR signals encoding the chemical shift values of  13 C α   i−1  and  15 N i  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13 C α ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in a direct time domain dimension, t 3 ( 1 H N ), and (2) to sine modulate the  13 C α   i−1  chemical shift evolution in t 1 ( 13 C α ) with the chemical shift evolution of  1 H α   i−1 ; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with an anti-phase peak pair derived from said sine modulating, wherein (1) the chemical shift values of  15 N i  and  1 H N   i  are measured in two frequency domain dimensions, ω 2 ( 15 N) and ω 3 ( 1 H N ), respectively, and (2) the chemical shift values of  1 H α   i−1  and  13 C α   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C α ), by the frequency difference between the two peaks forming said anti-phase peak pair and the frequency at the center of the two peaks, respectively, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift value of  1 H α   i−1  in a phase sensitive manner.   
   
   
       2 . The method according to  claim 1 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i−1  and  15 N i  in a phase sensitive manner in t 1 ( 13 C α ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ) and (2) to cosine modulate the  13 C α   i−1  chemical shift evolution in t 1 ( 13 C α ) with the chemical shift evolution of  1 H α   i−1  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with an in-phase peak pair derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       3 . The method according to  claim 1 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  15 N i  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with a peak pair wherein (1) the chemical shift value of  1 H N   i  is measured in a frequency domain dimension, ω 2 ( 1 H N ), and (2) the chemical shift values of  1 H α   i−1  and  13 C α   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C α ), by the frequency difference between the two peaks forming said peak pair and the frequency at the center of the two peaks, respectively. 
   
   
       4 . The method according to  claim 1 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 , occurs under conditions effective to generate NMR signals encoding the chemical shift value of  13 C′ i−1  in a phase sensitive manner in an indirect time domain dimension, t 4 ( 13 C′), and said processing the NMR signals generates a four dimensional (4D) NMR spectrum with a peak pair wherein (1) the chemical shift values of  15 N i ,  1 H N   i  and  13 C′ i−1  are measured in three frequency domain dimensions, ω 2 ( 15 N), ω 3 ( 1 H N ), and ω 4 ( 13 C′), respectively, and (2) the chemical shift values of  1 H α   i−1  and  13 C α   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C α ), by the frequency difference between the two peaks forming said peak pair and the frequency at the center of the two peaks, respectively. 
   
   
       5 . The method according to  claim 1 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i−1  and  15 N i  in a phase sensitive manner in t 1 ( 13 C α ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ), and (2) to avoid sine modulating the  13 C α   i−1  chemical shift evolution in t 1 ( 13 C α ) with the chemical shift evolution of  1 H α   i−1  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals generates a 3D NMR spectrum with an additional peak located centrally between two peaks forming said peak pair which measures the chemical shift value of  13 C α   i−1  along ω 1 ( 13 C α ). 
   
   
       6 . The method according to  claim 5 , wherein said additional peak is derived from  13 C α  nuclear spin polarization. 
   
   
       7 . The method according to  claim 6 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2B , wherein a radiofrequency pulse is used to create transverse  1 H α   i−1  magnetization, which is transferred to  13 C α   i−1 , to  15 N i , and to  1 H N   i , to generate the NMR signal. 
   
   
       8 . The method according to  claim 7 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3B  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3B , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pair and a second NMR subspectrum derived from said adding which contains said additional peak located centrally between the two peaks forming said peak pair.   
   
   
       9 . A method of conducting a reduced dimensionality three-dimensional (3D)  H , C ,-(C-TOCSY-CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) aliphatic protons of amino acid residue i−1,  1 H ali   i−1 ; (2) aliphatic carbons of amino acid residue i−1,  13 C ali   i−1 ; (3) a polypeptide backbone amide nitrogen of amino acid residue i,  15 N i ; and (4) a polypeptide backbone amide proton of amino acid residue i,  1 H N   i , wherein the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H ali   i−1  and  13 C ali   i−1  of amino acid residue i−1 are connected to the chemical shift evolutions of  15 N i  and  1 H N   i  of amino acid residue i, under conditions effective (1) to generate a NMR signal encoding the chemical shift values of  13 C ali   i−1  and  15 N i  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13 C ali ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in a direct time domain dimension, t 3 ( 1 H N ), and (2) to sine modulate the chemical shift evolutions of  13 C ali   i−1  in t 1 ( 13 C ali ) with the chemical shift evolutions of  1 H ali   i−1 ; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift values of  15 N i  and  1 H N   i  are measured in two frequency domain dimensions, ω 2 ( 15 N) and ω 3 ( 1 H N ), respectively, and (2) the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C ali ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs and the frequencies at the center of the two peaks, respectively, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift value of  1 H ali   i−1  in a phase sensitive manner.   
   
   
       10 . The method according to  claim 9 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C ali   i−1  and  15 N i  in a phase sensitive manner in t 1 ( 13 C ali ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ) and (2) to cosine modulate the chemical shift evolutions of  13 C ali   i−1  in t 1 ( 13 C ali ) with the chemical shift evolutions of  1 H ali   i−1  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       11 . The method according to  claim 9 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  15 N i  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with peak pairs wherein (1) the chemical shift value of  1 H N   i  is measured in a frequency domain dimension, ω 2 ( 1 H N ), and (2) the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C ali ), by the frequency differences between the two peaks forming said peak pairs and the frequencies at the center of the two peaks, respectively. 
   
   
       12 . The method according to  claim 9 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 , occurs under conditions effective to generate NMR signals encoding the chemical shift value of  13 C′ i−1  in a phase sensitive manner in an indirect time domain dimension, t 4 ( 13 C′), and said processing the NMR signals generates a four dimensional (4D) NMR spectrum with variant peak pairs wherein (1) the chemical shift values of  15 N i ,  1 H N   i  and  13 C′ i−1  are measured in three frequency domain dimensions, ω 2 ( 15 N), ω 3 ( 1 H N ), and ω 4 ( 13 C′), respectively, and (2) the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  are measured in a frequency domain dimension, ω 1 ( 13 C ali ), by the frequency differences between the two peaks forming said variant peak pairs and the frequencies at the center of the two peaks, respectively. 
   
   
       13 . The method according to  claim 9 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C ali   i−1  and  15 N i  in a phase sensitive manner in t 1 ( 13 C ali ) and t 2 ( 15 N) and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ), and (2) to avoid sine modulating the chemical shift evolutions of  13 C ali   i−1  in t 1 ( 13 C ali ) with the chemical shift evolution of  1 H α   i−1  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals generates a 3D NMR spectrum with additional peaks located centrally between said peak pairs which measure the chemical shift values of  13 C ali   i−1  along ω 1 ( 13 C ali ). 
   
   
       14 . The method according to  claim 13 , wherein said additional peaks are derived from  13 C ali  nuclear spin polarization. 
   
   
       15 . The method according to  claim 14 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2C , wherein a radiofrequency pulse is used to create transverse  1 H ali   i−1  magnetization, and  1 H ali   i−1  magnetization is transferred to  13 C ali   i−1 , to  13 C α   i−1 , to  13 C′ i−1 , to  15 N i , and to  1 H N   i , where the NMR signal is detected. 
   
   
       16 . The method according to  claim 15 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3C  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3C , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pairs, and a second NMR subspectrum derived from said adding which contains said additional peaks located centrally between said peak pairs.   
   
   
       17 . A method of conducting a reduced dimensionality three-dimensional (3D)  H   α/β   C   α/β ,CO,HA nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,  1 H β   i ; (2) a β-carbon of amino acid residue i,  13 C β   i ; (3) an α-proton of amino acid residue i,  1 H α   i ; (4) an α-carbon of amino acid residue i,  13 C α   i ; and (5) a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i , wherein the chemical shift values of  1 H α   i / 13 C α   i  and  1 H β   i / 13 C β   i  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H α   i ,  1 H β   i ,  13 C α   i , and  13 C β   i  are connected to the chemical shift evolution of  13 C′ i , under conditions effective (1) to generate NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  13 C′ i  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13 C α/β ) and t 2 ( 13 C′), respectively, and the chemical shift value of  1 H α   i  in a direct time domain dimension, t 3 ( 1 H α ) and (2) to sine modulate the chemical shift evolutions of  13 C α   i  and  13 C β   i  in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i , respectively; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift values of  13 C′ i  and  1 H α   i  are measured in two frequency domain dimensions, ω 2 ( 13 C′) and ω 3 ( 1 H α ), respectively, and (2) (i) the chemical shift values of  1 H α   i  and  1 H β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs, and (ii) the chemical shift values of  13 C α   i  and  13 C β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequencies at the center of the two peaks forming said anti-phase peak pairs, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift values of  1 H α   i  and  1 H β   i  in a phase sensitive manner.   
   
   
       18 . The method according to  claim 17 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  13 C′ i  in a phase sensitive manner in t 1 ( 13 C α/β ) and t 2 ( 13 C′), respectively, and the chemical shift value of  1 H α   i  in t 3 ( 1 H α ) and (2) to cosine modulate the  13 C α   i  and  13 C β   i  chemical shift evolutions in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i  for the additional NMR signals and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       19 . The method according to  claim 17 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  13 C′ i  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with peak pairs wherein (1) the chemical shift value of  1 H α   i  is measured in a frequency domain dimension, ω 2 ( 1 H α ), and (2) (i) the chemical shift values of  1 H α   i  and  1 H β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequency differences between two peaks forming said peak pairs, respectively, and (ii) the chemical shift values of  13 C α   i , and  13 C β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequencies at the center of the two peaks forming said peak pairs. 
   
   
       20 . The method according to  claim 17  wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  15 N i  in a phase sensitive manner in t 1 ( 13 C α/β ) and t 2 ( 15 N) and the chemical shift value of  1 H α   i  in t 3 ( 1 H α ), and (2) to avoid sine modulating the chemical shift evolutions of  13 C α   i  and  13 C β   i  in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i  for the additional NMR signal, and said processing the NMR signals and the additional NMR signals generates a 3D NMR spectrum with additional peaks located centrally between the two peaks forming said peak pairs which measure the chemical shift values of  13 C α   i  and  13 C β   i  along ω 1 ( 13 C α/β ). 
   
   
       21 . The method according to  claim 20 , wherein said additional peaks are derived from  13 C α  and  13 C β  nuclear spin polarization. 
   
   
       22 . The method according to  claim 21 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2E , wherein a radiofrequency pulse is used to create transverse  1 H α   i  and  1 H β   i  magnetization, and  1 H α   i  and  1 H β   i  polarization is transferred to  13 C α   i  and  13 C β   i , to  13 C′ i , and back to  1 H α   i , where the NMR signal is detected. 
   
   
       23 . The method according to  claim 22 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3E  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3E , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pairs, and a second NMR subspectrum derived from said adding which contains said additional peaks located centrally between the two peaks forming said peak pairs.   
   
   
       24 . A method of conducting a reduced dimensionality three-dimensional (3D)  H   α/β , C   α/β ,N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,  1 H β   i ; (2) a β-carbon of amino acid residue i,  13 C β   i ; (3) an α-proton of amino acid residue i,  1 H α   i ; (4) an α-carbon of amino acid residue i,  13 C α   i ; (5) a polypeptide backbone amide nitrogen of amino acid residue i,  15 N i ; and (6) a polypeptide backbone amide proton of amino acid residue i,  1 H N   i , wherein the chemical shift values of  1 H α   i / 13 C α   i  and  1 H β   i / 13 C β   i  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H α   i ,  1 H β   i ,  13 C α   i , and  13 C β   i  are connected to the chemical shift evolutions of  15 N i  and  1 H N   i , under conditions effective (1) to generate NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  15 N i  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13 C α/β ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in a direct time domain dimension, t 3 ( 1 H N ) and (2) to sine modulate the chemical shift evolutions of  13 C α   i  and  13 C β   i  in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i , respectively; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift values of  15 N i  and  1 H N   i  are measured in two frequency domain dimensions, ω 2 ( 15 N) and ω 3 ( 1 H N ), respectively, and (2) (i) the chemical shift values of  1 H α   i  and  1 H β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs, and (ii) the chemical shift values of  13 C α   i  and  13 C β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequencies at the center of said two peaks forming said anti-phase peak pairs, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift values of  1 H α   i  and  1 H β   i  in a phase sensitive manner.   
   
   
       25 . The method according to  claim 24 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  15 N i  in a phase sensitive manner in t 1 ( 13 C α/β ) and t 2 ( 15 N), respectively, and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ) and (2) to cosine modulate the  13 C α   i  and  13 C β   i  chemical shift evolutions in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       26 . The method according to  claim 24 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  15 N i  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with peak pairs wherein (1) the chemical shift value of  1 H N   i  is measured in a frequency domain dimension, ω 2 ( 1 H N ), and (2) (i) the chemical shift values of  1 H α   i  and  1 H β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequency differences between the two peaks forming said peak pairs, and (ii) the chemical shift values of  13 C α   i , and  13 C β   i  are measured in a frequency domain dimension, ω 1 ( 13 C α/β ), by the frequencies at the center of the two peaks forming said peak pairs. 
   
   
       27 . The method according to  claim 24 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C α   i ,  13 C β   i  and  15 N i  in a phase sensitive manner in t 1 ( 13 C α/β ) and t 2 ( 15 N) and the chemical shift value of  1 H N   i  in t 3 ( 1 H N ), and (2) to avoid sine modulating the chemical shift evolutions of  13 C α   i  and  13 C β   i  in t 1 ( 13 C α/β ) with the chemical shift evolutions of  1 H α   i  and  1 H β   i  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals generates a 3D NMR spectrum with additional peaks located centrally between the two peaks forming said peak pairs which measure the chemical shift values of  13 C α   i  and  13 C β   i  along ω 1 ( 13 C α/β ). 
   
   
       28 . The method according to  claim 27 , wherein said additional peaks are derived from  13 C α  and  13 C β  nuclear spin polarization. 
   
   
       29 . The method according to  claim 28 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2F , wherein a radiofrequency pulse is used to create transverse  1 H α   i  and  1 H β   i  magnetization, and  1 H α   i  and  1 H β   i  magnetization is transferred to  13 C α   i  and  13 C β   i , to  15 N i , and to  1 H N   i , where the NMR signal is detected. 
   
   
       30 . The method according to  claim 29 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3F  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3F , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pairs, and a second NMR subspectrum derived from said adding which contains said additional peaks located centrally between the two peaks forming said peak pairs.   
   
   
       31 . A method of conducting a reduced dimensionality three-dimensional (3D)  H , C ,C,H-COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for  1 H m ,  13 C m ,  1 H n , and  13 C n  of a protein molecule wherein m and n indicate atom numbers of two CH, CH 2  or CH 3  groups that are linked by a single covalent carbon-carbon bond in an amino acid residue, wherein the chemical shift values of  1 H m  and  13 C m  which are encoded in a peak pair of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H m  and  13 C m  are connected to the chemical shift evolutions of  1 H n  and  13 C m , under conditions effective (1) to generate NMR signals encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13 C m ) and t 2 ( 13 C n ), respectively, and the chemical shift value of  1 H n  in a direct time domain dimension, t 3 ( 1 H n ), and (2) to sine modulate the chemical shift evolution of  13 C m  in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m ; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift values of  13 C n  and  1 H n  are measured in two frequency domain dimensions, ω 2 ( 13 C n ) and ω 3 ( 1 H n ), respectively, and (2) the chemical shift values of  1 H m  and  13 C m  are measured in a frequency domain dimension, ω 1 ( 13 C m ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs and the frequencies at the center of the two peaks, respectively, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift value of  1 H m  in a phase sensitive manner.   
   
   
       32 . The method according to  claim 31 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in t 1 ( 13 C m ) and t 2 ( 13 C n ), respectively, and the chemical shift value of  1 H n  in t 3 ( 1 H n ), and (2) to cosine modulate the  13 C m  chemical shift evolution in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       33 . The method according to  claim 31 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  13 C n  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with peak pairs wherein (1) the chemical shift value of  1 H n  is measured in a frequency domain dimension, ω 2 ( 1 H n ), and (2) the chemical shift values of  1 H m  and  13 C m  are measured in a frequency domain dimension, ω 1 ( 13 C m ), by the frequency differences between the two peaks forming said peak pairs and the frequencies at the center of the two peaks, respectively. 
   
   
       34 . The method according to  claim 31 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in t 1 ( 13 C m ) and t 2 ( 13 C n ) and the chemical shift value of  1 H n  in t 3 ( 1 H), and (2) to avoid sine modulating the chemical shift evolution of  13 C m  in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m  for the additional NMR signal, and said processing the NMR signals and the additional NMR signal generates a 3D NMR spectrum with additional peaks located centrally between the two peaks forming said peak pairs which measure the chemical shift value of  13 C m  along ω 1 ( 13 C m ). 
   
   
       35 . The method according to  claim 34 , wherein said additional peaks are derived from  13 C m  nuclear spin polarization. 
   
   
       36 . The method according to  claim 35 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2H , wherein a radiofrequency pulse is used to create transverse  1 H m  magnetization, and  1 H m  magnetization is transferred to  13 C m , to  13 C n , and to  1 H n , where the NMR signal is detected. 
   
   
       37 . The method according to  claim 36 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3H  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3H , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pairs, and a second NMR subspectrum derived from said adding which contains said additional peaks located centrally between the two peaks forming said peak pairs.   
   
   
       38 . A method of conducting a reduced dimensionality three-dimensional (3D)  H , C ,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for  1 H m ,  13 C m ,  1 H n , and  13 C n  of a protein molecule wherein m and n indicate atom numbers of two CH, CH 2  or CH 3  groups that may or may not be directly linked by a single covalent carbon-carbon bond in an amino acid residue, wherein the chemical shift values of  1 H m  and  13 C m  which are encoded in a peak pair of a 3D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H m  and  13 C m  are connected to the chemical shift evolutions of  1 H n  and  13 C n , under conditions effective (1) to generate NMR signals encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in two indirect time domain dimensions, t 1 ( 13  C m ) and t 2 ( 13 C n ), respectively, and the chemical shift value of  1 H n  in a direct time domain dimension, t 3 ( 1 H n ), and (2) to sine modulate the chemical shift evolution of  13 C m  in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m ; and   processing the NMR signals to generate a sine-modulated 3D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift values of  13 C n  and  1 H n  are measured in two frequency domain dimensions, ω 2 ( 13 C n ) and ω 3 ( 1 H n ), respectively, and (2) the chemical shift values of  1 H m  and  13 C m  are measured in a frequency domain dimension, ω 1 ( 13 C m ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs and the frequencies at the center of the two peaks, respectively, wherein said sine-modulated 3D NMR spectrum enables detection of the chemical shift value of  1 H m  and in a phase sensitive manner.   
   
   
       39 . The method according to  claim 38 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in t 1 ( 13 C m ) and t 2 ( 13 C n ), respectively, and the chemical shift value of  1 H n  in t 3 ( 1 H n ) and (2) to cosine modulate the  13 C m  chemical shift evolution in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 3D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR spectrum and said cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum generated by subtracting said cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR spectrum, wherein combined use of said sum 3D NMR spectrum and said difference 3D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       40 . The method according to  claim 38 , wherein said applying radiofrequency pulses is carried out so that the chemical shift evolution of  13 C n  does not occur and said processing the NMR signals generates a two dimensional (2D) NMR spectrum with peak pairs wherein (1) the chemical shift value of  1 H n  is measured in a frequency domain dimension, ω 2 ( 1 H n ), and (2) the chemical shift values of  1 H m  and  13 C m  are measured in a frequency domain dimension, ω 1 ( 13 C m ), by the frequency differences between the two peaks forming said peak pairs and the frequencies at the center of the two peaks, respectively. 
   
   
       41 . The method according to  claim 38 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift values of  13 C m  and  13 C n  in a phase sensitive manner in t 1 ( 13 C m ) and t 2 ( 13 C n ) and the chemical shift value of  1 H n  in t 3 ( 1 H n ), and (2) to avoid sine modulating the chemical shift evolution of  13 C m  in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals generates a 3D NMR spectrum with additional peaks located centrally between the two peaks forming said peak pairs which measure the chemical shift value of  13 C m  along ω 1 ( 13 C m ). 
   
   
       42 . The method according to  claim 41 , wherein said additional peaks are derived from  13 C m  nuclear spin polarization. 
   
   
       43 . The method according to  claim 42 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2I , wherein a radiofrequency pulse is used to create transverse  1 H m  magnetization, and  1 H m  magnetization is transferred to  13 C m , to  13 C n , and to  1 H n , where the NMR signal is detected. 
   
   
       44 . The method according to  claim 43 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3I  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3I , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pairs, and a second NMR subspectrum derived from said adding which contains said additional peaks located centrally between the two peaks forming said peak pairs.   
   
   
       45 . A method of conducting a reduced dimensionality two-dimensional (2D)  HB , CB ,(CG,CD),HD nuclear magnetic resonance NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule: (1) a β-proton of an amino acid residue with an aromatic side chain,  1 H β ; (2) a β-carbon of an amino acid residue with an aromatic side chain,  13 C β ; and (3) a δ-proton of an amino acid residue with an aromatic side chain,  1 H δ , wherein the chemical shift values of  1 H β  and  13 C β  which are encoded in a peak pair of a 2D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H β  and  13 C β  are connected to the chemical shift evolution of  1 H δ , under conditions effective (1) to generate NMR signals encoding the chemical shift value of  13 C β  in a phase sensitive manner in an indirect time domain dimension, t 1 ( 13 C β ), and the chemical shift value of  1 H δ  in a direct time domain dimension, t 2 ( 1 H δ ), and (2) to sine modulate the chemical shift evolution of  13 C β  in t 1 ( 13 C β ) with the chemical shift evolution of  1 H β ; and   processing the NMR signals to generate a sine-modulated 2D NMR spectrum with an anti-phase peak pair derived from said sine modulating wherein (1) the chemical shift value of  1 H δ  is measured in a frequency domain dimension, ω 2 ( 1 H δ ), and (2) the chemical shift values of  1 H β  and  13 C β  are measured in a frequency domain dimension, ω 1 ( 13 C β ), by the frequency difference between the two peaks forming said anti-phase peak pair and the frequency at the center of the two peaks, respectively, wherein said sine-modulated 2D NMR spectrum enables detection of the chemical shift value of  1 H β  in a phase sensitive manner.   
   
   
       46 . The method according to  claim 45 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift value of  13 C β  in a phase sensitive manner in t 1 ( 13 C β ) and the chemical shift value of  1 H δ  in t 2 ( 1 H δ ) and (2) to cosine modulate the  13 C β  chemical shift evolution in t 1 ( 13 C β ) with the chemical shift evolution of  1 H β  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 2D NMR spectrum with an in-phase peak pair derived from said cosine modulating, a sum 2D NMR spectrum generated by adding said sine-modulated 2D NMR spectrum and said cosine-modulated 2D NMR spectrum, and a difference 2D NMR spectrum generated by subtracting said cosine-modulated 2D NMR spectrum from said sine-modulated 2D NMR spectrum, wherein combined use of said sum 2D NMR spectrum and said difference 2D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       47 . The method according to  claim 45 , wherein said applying radiofrequency pulses is carried out so that:
 (i) the chemical shift evolution of a δ-carbon of an amino acid residue with an aromatic side chain,  13 C δ , occurs under conditions effective to generate NMR signals encoding the chemical shift value of  13 C δ  in a phase sensitive manner in an indirect time domain dimension, t 3 ( 13 C δ ), and said processing the NMR signals generates a three dimensional (3D) NMR spectrum with a peak pair wherein (1) the chemical shift values of  1 H δ  and  13 C δ  are measured in two frequency domain dimensions, ω 2 ( 1 H δ ) and ω 3 ( 13 C δ ), respectively, and (2) the chemical shift values of  1 H β  and  13 C β  are measured in a frequency domain dimension, ω 1 ( 13 C β ), by the frequency difference between the two peaks forming said peak pair and the frequency at the center of the two peaks, respectively; or   (ii) the chemical shift evolution of a γ-carbon of an amino acid residue with an aromatic side chain,  13 C γ  occurs under conditions effective to generate NMR signals encoding the chemical shift value of  13 C γ , in a phase sensitive manner in an indirect time domain dimension, t 3 ( 13 C γ ), and said processing the NMR signals generates a three dimensional (3D) NMR spectrum with a peak pair wherein (1) the chemical shift values of  1 H δ  and  13 C γ  are measured in two frequency domain dimensions, ω 2 ( 1 H δ ) and ω 3 ( 13 C γ ), respectively, and (2) the chemical shift values of  1 H β  and  13 C β  are measured in a frequency domain dimension, ω 1 ( 13 C β ), by the frequency difference between the two peaks forming said peak pair and the frequency at the center of the two peaks, respectively.   
   
   
       48 . The method according to  claim 46 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift value of  13 C β  in a phase sensitive manner in t 1 ( 13 C β ) and the chemical shift value of  1 H δ  in t 2 ( 1 H δ ), and (2) to avoid sine modulating the chemical shift evolution of  13 C β  in t 1 ( 13 C β ) with the chemical shift evolution of  1 H β  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals generates a 2D NMR spectrum with an additional peak located centrally between said peak pair which measure the chemical shift value of  13 C β  along ω 1 ( 13 C β ). 
   
   
       49 . The method according to  claim 48 , wherein said additional peak is derived from  13 C β  nuclear spin polarization. 
   
   
       50 . The method according to  claim 49 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2J , wherein a radiofrequency pulse is used to create transverse  1 H β  magnetization, and  1 H β  magnetization is transferred to  13 C β , to  13 C δ , and to  1 H δ , where the NMR signal is detected. 
   
   
       51 . The method according to  claim 50 , wherein said applying radiofrequency pulses comprises:
 applying a first set of radiofrequency pulses according to the scheme shown in  FIG. 3J  to generate a first NMR signal, and   applying a second set of radiofrequency pulses according to the scheme shown in  FIG. 3J , wherein phase φ 1  of the first  1 H pulse is altered by 180° to generate a second NMR signal, said method further comprising:   adding and subtracting the first NMR signal and the second NMR signal prior to said processing, whereby said processing the NMR signals generates a first NMR subspectrum derived from said subtracting which contains said peak pair, and a second NMR subspectrum derived from said adding which contains said additional peak located centrally between the two peaks forming said peak pair.   
   
   
       52 . A method of conducting a reduced dimensionality two-dimensional (2D)  H , C ,H-COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for  1 H m ,  13 C m , and  1 H n  of a protein molecule wherein m and n indicate atom numbers of two CH, CH 2  or CH 3  groups in an amino acid residue, wherein the chemical shift values of  1 H m  and  13 C m  which are encoded in a peak pair of a 2D NMR spectrum are detected in a phase sensitive manner, said method comprising:
 providing a protein sample;   applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer wherein the chemical shift evolutions of  1 H m  and  13 C m  are connected to the chemical shift evolution of  1 H n , under conditions effective (1) to generate NMR signals encoding the chemical shift value of  13 C m  in a phase sensitive manner in an indirect time domain dimension, t 1 ( 13 C m ), and the chemical shift value of  1 H n  in a direct time domain dimension, t 2 ( 1 H n ) and (2) to sine modulate the chemical shift evolution of  13 C m  in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m ; and   processing the NMR signals to generate a sine-modulated 2D NMR spectrum with anti-phase peak pairs derived from said sine modulating wherein (1) the chemical shift value of  1 H n  is measured in a frequency domain dimension, ω 2 ( 1 H n ), and (2) the chemical shift values of  1 H m  and  13 C m  are measured in a frequency domain dimension, ω 1 ( 13 C m ), by the frequency differences between each of the two peaks forming each of said anti-phase peak pairs and the frequencies at the center of the two peaks, respectively, wherein said sine-modulated 2D NMR spectrum enables detection of the chemical shift value of  1 H m  in a phase sensitive manner.   
   
   
       53 . The method according to  claim 52 , wherein said applying radiofrequency pulses is carried out under conditions effective (1) to generate additional NMR signals encoding the chemical shift value of  13 C m  in a phase sensitive manner in t 1 ( 13 C m ) and the chemical shift value of  1 H n  in t 2 ( 13 H n ) and (2) to cosine modulate the  13 C m  chemical shift evolution in t 1 ( 13 C m ) with the chemical shift evolution of  1 H m  for the additional NMR signals, and said processing the NMR signals and the additional NMR signals further comprises generating a cosine-modulated 2D NMR spectrum with in-phase peak pairs derived from said cosine modulating, a sum 2D NMR spectrum generated by adding said sine-modulated 2D NMR spectrum and said cosine-modulated 2D NMR spectrum, and a difference 2D NMR spectrum generated by subtracting said cosine-modulated 2D NMR spectrum from said sine-modulated 2D NMR spectrum, wherein combined use of said sum 2D NMR spectrum and said difference 2D NMR spectrum enables placement of the two peaks forming said peak pairs into separate spectra, thereby allowing phase-sensitive editing of the two peaks forming said peak pairs. 
   
   
       54 . The method according to  claim 52 , wherein said applying radiofrequency pulses effects a nuclear spin polarization transfer according to  FIG. 2K , wherein a radiofrequency pulse is used to create transverse  1 H m  magnetization, and  1 H m  polarization is transferred to  13 C m , to  1 H m , and to  1 H n , where the NMR signal is detected. 
   
   
       55 . The method according to  claim 54 , wherein said applying radiofrequency pulses is carried out according to the scheme shown in  FIG. 3K . 
   
   
       56 . A method for sequentially assigning chemical shift values of an α-proton,  1 H α , an α-carbon,  13 C α , a polypeptide backbone amide nitrogen,  15 N, and a polypeptide backbone amide proton,  1 H N , of a protein molecule comprising:
 providing a protein sample;   conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein the chemical shift values of  1 H α  and  13 C α  which are encoded in a peak pair of a 3D NMR spectrum are detected in a phase sensitive manner, comprising: (1) a RD three dimensional (3D)  HA , CA ,(CO),N,HN NMR experiment to measure and connect chemical shift values of the α-proton of amino acid residue i−1,  1 H α   i−1 , the α-carbon of amino acid residue i−1,  13 C α   i−1 , the polypeptide backbone amide nitrogen of amino acid residue i,  15 N i , and the polypeptide backbone amide proton of amino acid residue i,  1 H N   i  and (2) a RD 3D HNN CAHA  NMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H α ,  13 C α ,  15 N, and  1 H N  by (i) matching the chemical shift values of  1 H α   i−1  and  13 C α   i−1  with the chemical shift values of  1 H α   i  and  13 C α   i , (ii) using the chemical shift values of  1 H α   i−1  and  13 C α   i−1  to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.   
   
   
       57 . The method according to  claim 56  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β (CO)NUN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i−1,  1 H β   i−1 , the β-carbon of amino acid residue i−1,  13 C β   i−1 ,  1 H α   i−1 ,  13 C α   i−1 ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H β  and  13 C β  by using the chemical shift values of  1 H β   i−1  and  13 C β   i−1  to identify the type of amino acid residue i−1.   
   
   
       58 . The method according to  claim 57  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β , C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,  1 H β   i , the β-carbon of amino acid residue i,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i  by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H   α/β , C   α/β ,CO,HA NMR experiment with the sequentially assigned chemical shift values of  1 H β ,  13 C β ,  1 H α ,  13 C α ,  15 N, and  1 H N  measured by said RD 3D  HA , CA ,(CO),N,HN NMR experiment, RD 3D HNN CAHA  NMR experiment, and RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment.   
   
   
       59 . The method according to  claim 57  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β , C   α/β ,N,HN NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  with the chemical shift values of  1 H β   i−1 ,  13 C β   i−1 ,  1 H α   i−1 , and  13 C α   i−1  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment.   
   
   
       60 . The method according to  claim 57  further comprising:
 subjecting the protein sample to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of  13 C β   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  13 C β   i  and  13 C α   i  measure by said 3D HNNCACB NMR experiment with the chemical shift values of  13 C β   i−1  and  13 C α   i−1  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment.   
   
   
       61 . The method according to  claim 57  further comprising:
 subjecting the protein sample to a RD two-dimensional (2D)  HB , CB ,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of  1 H β   i−1 ,  13 C β   i−1 , and a δ-proton of amino acid residue i−1 with an aromatic side chain,  1 H δ   i−1 ; and   obtaining sequential assignments by (i) matching the chemical shift values of  1 H β   i−1  and  13 C β   i−1  measured by said RD 2D  HB ,  CB , (CG,CD), HD NMR experiment with the chemical shift values of  1 H β  and  13 C β  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment, (ii) using said chemical shift values to identify amino acid residue i as having an aromatic side chain, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along said polypeptide chain.   
   
   
       62 . The method according to  claim 57  further comprising:
 subjecting the protein sample to a RD 3D  H , C ,C,H-COSY NMR experiment or a RD 3D  H , C ,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,  1 H ali   i , and aliphatic carbons of amino acid residue i,  13 C ali   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i  and  13 C ali   i , by (i) matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured using said RD 3D  H , C ,C,H-COSY NMR experiment or RD 3D  H , C ,C,H-TOCSY RD NMR experiment with the chemical shift values of  1 H β ,  13 C β ,  1 H α   i , and  13 C α   i  measured by said RD 3D  HA , CA ,(CO),N,HN NMR experiment, RD 3D HNN CAHA  NMR experiment, and RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and (ii) using the chemical shift values of  1 H ali   i  and  13 C ali   i , to identify the type of amino acid residue i.   
   
   
       63 . The method according to  claim 56  further comprising:
 subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i−1  by matching the chemical shift value of  13 C α   i  measured by said RD 3D HNN< CO , CA > NMR experiment with the sequentially assigned chemical shift values of  13 C α ,  15 N, and  1 H N  measured by said RD 3D  HA , CA ,(CO),N,HN NMR experiment and RD 3D HNN CAHA  NMR experiment.   
   
   
       64 . The method according to  claim 56  further comprising:
 subjecting the protein sample to (i) a RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,  1 H β   i , the β-carbon of amino acid residue i,  13 C β   i , the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i  and (ii) a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of  13 C′ i , the α-carbon of amino acid residue i+1,  13 C α   i+1 , the polypeptide backbone amide nitrogen of amino acid residue i+1,  15 N i+1 , and the polypeptide backbone amide proton of amino acid residue i+1,  1 H N   i+1 ; and   obtaining sequential assignments by matching the chemical shift value of  13 C′ i  measured by said RD 3D HNN< CO , CA > NMR experiment with the chemical shift value of  13 C′ i  measured by said RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment.   
   
   
       65 . The method according to  claim 56 , further comprising:
 subjecting the protein sample to a RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i−1,  1 H ali   i−1 , aliphatic carbons of amino acid residue i−1,  13 C ali   i−1 ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  for amino acid residues i having unique pairs of  15 N i  and  1 H N   i  chemical shift values by matching the chemical shift values of  1 H α  and  13 C α  measured by said RD 3D HNN CAHA  NMR experiment and RD 3D  HA , CA ,(CO),N,HN NMR experiment with the chemical shift values of  1 H α   i−1  and  13 C α   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and using the  1 H ali   i−1  and  13 C ali   i−1  chemical shift values to identify the type of amino acid residue i−1.   
   
   
       66 . The method according to  claim 56  further comprising:
 subjecting the protein sample to a RD 3D  H , C ,C,H-COSY NMR experiment or a RD 3D  H , C ,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,  1 H ali   i , and aliphatic carbons of amino acid residue i,  13 C ali   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i  and  13 C ali   i  by (i) matching the chemical shift values of  1 H α   i  and  13 C α   i  measured using said RD 3D  H , C ,C,H-COSY NMR experiment or RD 3D  H , C ,C,H-TOCSY RD NMR experiment with the chemical shift values of  1 H α  and  13 C α  measured by said RD 3D  HA , CA ,(CO),N,HN NMR experiment and RD 3D HNN CAHA  NMR experiment and (ii) using the chemical shift values of  1 H ali   i  and  13 C ali   i , to identify the type of amino acid residue i.   
   
   
       67 . A method for sequentially assigning chemical shift values of a β-proton,  1 H β , a β-carbon,  13 C β , an α-proton,  1 H α , an α-carbon,  13 C α , a polypeptide backbone amide nitrogen,  15 N, and a polypeptide backbone amide proton,  1 H N , of a protein molecule comprising:
 providing a protein sample;   conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein the chemical shift values of  1 H α/β  and  13 C α/β  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, comprising: (1) a RD three-dimensional (3D)  H   α/β   C   α/β (CO)NHN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i−1,  1 H β   1−i , the β-carbon of amino acid residue i−1,  13 C β   i−1 , the α-proton of amino acid residue i−1,  1 H α   i−1 , the α-carbon of amino acid residue i−1,  13 C α   i−1 , the polypeptide backbone amide nitrogen of amino acid residue i,  15 N i , and the polypeptide backbone amide proton of amino acid residue i,  1 H N   i  and (2) a RD 3D  H   α/β , C   α/β ,N,HN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,  1 H β   i , the β-carbon of amino acid residue i,  13 C β   i , the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H β ,  13 C β ,  1 H α ,  13 C α ,  15 N, and  1 H N  by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,  1 H α/β   i−1 , and the α- and β-carbons of amino acid residue i−1,  13 C α/β   i−1 , with the chemical shift values of  1 H α/β   i  and  13 C α/β   i , (ii) using the chemical shift values of  1 H α/β   i−1  and  13 C α/β   i , to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.   
   
   
       68 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a RD 3D  HA , CA ,(CO),N,HN NMR experiment (i) to measure and connect chemical shift values of  1 H α   i−1 ,  13 C α   i−1 ,  15 N i , and  1 H N   i  and (ii) to distinguish between NMR signals for  1 H α / 13 C α  and  1 H β / 13 C β  measured in said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β , C   α/β N,HN NMR experiment.   
   
   
       69 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i  by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H   α/β , C   α/β ,CO,HA NMR experiment with the sequentially assigned chemical shift values of  1 H β ,  13 C β ,  1 H α ,  13 C α ,  15 N, and  1 H N  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β , C   α/β ,N,HN NMR experiment.   
   
   
       70 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 ,  13 C′ i−1 ,  13 C α   i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i−1  by matching the chemical shift value of  13 C α   i  measured by said RD 3D HNN< CO , CA > NMR experiment with the sequentially assigned chemical shift values of  13 C α ,  15 N, and  1 H N  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β , C   α/β ,N,HN NMR experiment.   
   
   
       71 . The method according to  claim 67  further comprising:
 subjecting the protein sample to (i) a RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i  and (ii) a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of  13 C′ i , the α-carbon of amino acid residue i+1,  13 C α   i+1 , the polypeptide backbone amide nitrogen of amino acid residue i+1,  15 N i+1 , and the polypeptide backbone amide proton of amino acid residue i+1,  1 H N   i+1 ; and   obtaining sequential assignments by matching the chemical shift value of  13 C′ i  measured by said RD 3D HNN< CO , CA > NMR experiment with the chemical shift value of  13 C′ i  measured by said RD 3D  H   α/β , C   α/β ,CO,HA NMR experiment.   
   
   
       72 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemical shift values of  1 H ali   i−1 ,  13 C ali   i−1 ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  for amino acid residues i having unique pairs of  15 N i  and  1 H N   i  chemical shift values by matching the chemical shift values of  1 H β ,  13 C β   ,    1 H α , and  13 C α  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β , C   α/β ,N,HN NMR experiment with the chemical shift values of  1 H β   i−1 ,  13 C β   i−1 ,  1 H α   i−1 , and  13 C α   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and using the  1 H ali   i−1  and  13 C ali   i−1  chemical shift values to identify the type of amino acid residue i−1.   
   
   
       73 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of  13 C β   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  13 C β   i  and  13 C α   i  measured by said 3D HNNCACB NMR experiment with the chemical shift values of  13 C β   i−1  and  13 C α   i−1  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment.   
   
   
       74 . The method according to  claim 67  further comprising:
 subjecting the protein sample to a RD two-dimensional (2D)  HB , CB ,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i , and a δ-proton of amino acid residue i with an aromatic side chain,  1 H δ   i ; and   obtaining sequential assignments by (i) matching the chemical shift values of  1 H β   i  and  13 C β   i  measured by said RD 2D  HC , CB , (CG, CD), HDNMR experiment with the chemical shift values of  1 H β  and  13 C β  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment, (ii) using said chemical shift values to identify amino acid residue i as having an aromatic side chain, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along said polypeptide chain.   
   
   
       75 . The method according to  claim 67 , further comprising:
 subjecting the protein sample to a RD 3D  H , C ,C,H-COSY NMR experiment or a RD 3D  H , C ,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,  1 H ali  i and aliphatic carbons of amino acid residue i,  13 C ali  i; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i  and  13 C ali   i  by (i) matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured using said RD 3D  H , C ,C,H-COSY NMR experiment or RD 3D  H , C ,C,H-TOCSY RD NMR experiment with the chemical shift values of  1 H β ,  13 C β ,  1 H α , and  13 C α  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment, and (ii) using the chemical shift values of  1 H ali   i  and  13 C ali   i , to identify the type of amino acid residue i.   
   
   
       76 . A method for sequentially assigning the chemical shift values of aliphatic protons,  1 H ali , aliphatic carbons,  13 C ali , a polypeptide backbone amide nitrogen,  15 N, and a polypeptide backbone amide proton,  1 H N , of a protein molecule comprising:
 providing a protein sample;   conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein the chemical shift values of  1 H ali  and  13 C ali  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, comprising: (1) a RD three-dimensional (3D)  H , C ,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,  1 H ali   i−1 , the aliphatic carbons of amino acid residue i−1,  13 C ali   i−1 , the polypeptide backbone amide nitrogen of amino acid residue i,  15 N i , and the polypeptide backbone amide proton of amino acid residue i,  1 H N   i  and (2) a RD 3D  H   α/β   C   α/β ,N,HN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,  1 H β   i , the β-carbon of amino acid residue i,  13 C β   i , the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali ,  13 C ali ,  15 N, and  1 H N  by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,  1 H α/β   i−1  and the α- and β-carbons of amino acid residue i−1,  13 C α/β   i−1  with the chemical shift values of  1 H α/β   i  and  13 C α/β   i  of amino acid residue i, (ii) using the chemical shift values of  1 H ali   i−i  and  13 C ali   i−1  to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.   
   
   
       77 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β , CO,HA NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i  by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment with the sequentially assigned chemical shift values of  1 H β ,  13 C β ,  1 H α ,  13 C α ,  15 N, and  1 H N  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment.   
   
   
       78 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i−1  by matching the chemical shift value of  13 C α   i  measured by said RD 3D HNN< CO , CA > NMR experiment with the sequentially assigned chemical shift values of  13 C α ,  15 N, and  1 H N  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment.   
   
   
       79 . The method according to  claim 76  further comprising:
 subjecting the protein sample to (i) a RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i  and (ii) a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of  13 C′ i , the α-carbon of amino acid residue i+1,  13 C α   i+1 , the polypeptide backbone amide nitrogen of amino acid residue i+1,  15 N i+1 , and the polypeptide backbone amide proton of amino acid residue i+1,  1 H N   i+1 ; and   obtaining sequential assignments by matching the chemical shift value of  13 C′ i  measured by said RD 3D HNN< CO , CA > NMR experiment with the chemical shift value of  13 C′ i  measured by said RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment.   
   
   
       80 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment (i) to measure and connect the chemical shift values of  1 H α/β   i−1 ,  13 C α/β   i−1 ,  15 N i , and  1 H N   i , and (ii) to identify NMR signals for  1 H α/β   i−1 ,  13 C α/β   i−1 ,  15 N i , and  1 H N   i  in said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment.   
   
   
       81 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD 3D  HA , CA ,(CO),N,HN NMR experiment (i) to measure and connect chemical shift values of  1 H α   i−1 ,  13 C α   i−1 ,  15 N i , and  1 H N   i  and (ii) to identify NMR signals for  1 H α  and  13 C α  in said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment.   
   
   
       82 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of  13 C β   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  13 C β   i  and  13 C α   i  measured by said 3D HNNCACB NMR experiment with the chemical shift values of  13 C β   i−1  and  13 C α   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment.   
   
   
       83 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD two-dimensional (2D)  HB , CB ,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i , and a δ-proton of amino acid residue i with an aromatic side chain,  1 H δ   i ; and   obtaining sequential assignments by matching the chemical shift values of  1 H β   i  and  13 C β   i  measured by said RD 2D  HB , CB , (CG, CD), HD NMR experiment with the chemical shift values of  1 H β  and  13 C β  measured by said RD 3D  H   α/β   C   α/β ,N,HN NMR experiment and RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment, using said chemical shift values to identify amino acid residue i as having an aromatic side chain, and mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along said polypeptide chain.   
   
   
       84 . The method according to  claim 76  further comprising:
 subjecting the protein sample to a RD 3D  H , C ,C,H-COSY NMR experiment or a RD 3D  H , C ,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,  1 H ali   i  and aliphatic carbons of amino acid residue i,  13 C ali   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i  and  13 C ali   i  by (i) matching the chemical shift values of  1 H ali   i  and  13 C ali   i  measured using said RD 3D  H , C ,C,H-COSY NMR experiment or RD 3D  H , C ,C,H-TOCSY NMR experiment with the chemical shift values of  1 H ali  and  13 C ali  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D  H   α/β   C   α/β ,N,HN NMR experiment, and (ii) using the chemical shift values of  1 H ali   i  and  13 C ali   i , to identify the type of amino acid residue i.   
   
   
       85 . A method for sequentially assigning chemical shift values of aliphatic protons,  1 H ali , aliphatic carbons,  13 C ali , a polypeptide backbone amide nitrogen,  15 N, and a polypeptide backbone amide proton,  1 H N , of a protein molecule comprising:
 providing a protein sample;   conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein the chemical shift values of  1 H ali  and  13 C ali  which are encoded in peak pairs of a 3D NMR spectrum are detected in a phase sensitive manner, comprising: (1) a RD three-dimensional (3D)  H , C ,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,  1 H ali   i−1 , the aliphatic carbons of amino acid residue i−1,  13 C ali   i−1 , the polypeptide backbone amide nitrogen of amino acid residue i,  15 N i , and the polypeptide backbone amide proton of amino acid residue i,  1 H N   i  and (2) a RD 3D HNN CAHA  NMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali ,  13 C ali ,  15 N, and  1 H N  by (i) matching the chemical shift values of the α-proton of amino acid residue i−1,  1 H α   i−1 , and the α-carbon of amino acid residue i−1,  13 C α   i−1 , with the chemical shift values of  1 H α   i  and  13 C α   i , (ii) using the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.   
   
   
       86 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β , CO,HA NMR experiment to measure and connect the chemical shift values of a β-proton of amino acid residue i,  1 H β   i , a β-carbon of amino acid residue i,  13 C β   i ,  1 H α   i ,  13 C α   i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i  by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment with the sequentially assigned chemical shift values of  1 H β ,  13 C β ,  1 H α ,  13 C β ,  15 N, and  1 H N  measured by said RD 3D H C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNN CAHA  NMR experiment.   
   
   
       87 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,  13 C′ i−1 ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments of the chemical shift value of  13 C′ i−1  by matching the chemical shift value of  13 C α   i  measured by said RD 3D HNN< CO , CA > NMR experiment with the sequentially assigned chemical shift values of  13 C α ,  15 N, and  1 H N  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNN CAHA  NMR experiment.   
   
   
       88 . The method according to  claim 85  further comprising:
 subjecting the protein sample to (i) a RD 3D  H   α/β   C   α/β ,CO,HA NMR experiment to measure and connect the chemical shift values of a β-proton of amino acid residue i,  1 H β   i , a β-carbon of amino acid residue i,  13 C β   i , the α-proton of amino acid residue i,  1 H α   i , the α-carbon of amino acid residue i,  13 Cα i , and a polypeptide backbone carbonyl carbon of amino acid residue i,  13 C′ i  and (ii) a RD 3D HNN< CO , CA > NMR experiment to measure and connect the chemical shift values of  13 C′ i , an α-carbon of amino acid residue i+1,  13 C α   i+1 , a polypeptide backbone amide nitrogen of amino acid residue i+1,  15 N i+1 , and a polypeptide backbone amide proton of amino acid residue i+1,  1 H N   i+1 ; and   obtaining sequential assignments by matching the chemical shift value of  13 C′ i  measured by said RD 3D HNN< CO , CA > NMR experiment with the chemical shift value of  13 C′ i  measured by said RD 3D  H   α/β , C   α/β ,CO,HA NMR experiment.   
   
   
       89 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β   C   α/β (CO)NUN NMR experiment (i) to measure and connect the chemical shift values of the α- and β-protons of amino acid residue i−1,  1 H α/β   i−1 , α- and β-carbons of amino acid residue i−1,  13 C α/β   i−1 ,  15 N i , and  1 H N   i , and (ii) to distinguish NMR signals for the chemical shift values of  1 H β   i−1 ,  13 C β   i−1 ,  1 H α   i−1 , and  13 C α   i−1  measured by said RD 3D  H   α/β   C   α/β (CO)NHN NMR experiment from NMR signals for the chemical shift values of  1 H ali   i−1  and  13 C ali   i−1  other than  1 H α/β   i−1  and  13 C α/β   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment.   
   
   
       90 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD 3D  H   α/β , C   α/β ,N,HN NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H   α/β   C   α/β ,N,HN NMR experiment with the chemical shift values of  1 H β   i−1 ,  13 C β   i−1 ,  1 H α   i−1 , and  13 C α   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment.   
   
   
       91 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a 3D HNNCACB NMR experiment to measure and connect the chemical shift values of  13 C β   i ,  13 C α   i ,  15 N i , and  1 H N   i ; and   obtaining sequential assignments by matching the chemical shift values of  13 C β   i  and  13 C α   i  measured by said 3D HNNCACB NMR experiment with the chemical shift values of  13 C β   i−1  and  13 C α   i−1  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment.   
   
   
       92 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD two-dimensional (2D)  HB , CB ,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of  1 H β   i ,  13 C β   i , and a δ-proton of amino acid residue i with an aromatic side chain,  1 H δ   i ; and   obtaining sequential assignments by matching the chemical shift values of  1 H β   i  and  13 C β   i  measured by said RD 2D  HB ,  CB ,(CG, CD), HD NMR experiment with the chemical shift values of  1 H β  and  13 C β  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment, using said chemical shift values to identify amino acid residue i as having an aromatic side chain, and mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along said polypeptide chain.   
   
   
       93 . The method according to  claim 85  further comprising:
 subjecting the protein sample to a RD 3D  H , C ,C,H-COSY NMR experiment or a RD 3D  H , C ,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,  1 H ali   i  and aliphatic carbons of amino acid residue i,  13 C ali   i ; and   obtaining sequential assignments of the chemical shift values of  1 H ali   i  and  13 C ali   i  by (i) matching the chemical shift values of  1 H ali  and  13 C ali  measured using said RD 3D  H , C ,C,H-COSY NMR experiment or RD 3D  H , C ,C,H-TOCSY NMR experiment with the chemical shift values of  1 H β   i ,  13 C β   i ,  1 H α   i , and  13 C α   i  measured by said RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNN CAHA  NMR experiment, and (ii) using the chemical shift values of  1 H ali   i  and  13 C ali   i , to identify the type of amino acid residue i.