Sea-trosy and related methods

Abstract

A method for preferentially observing an exposed position ( 1 c ) of a macromolecule. A sample is obtained having a macromolecule ( 1 a ) with a first proton ( 1 ) and a second molecule ( 2 a ) with a second proton ( 2 ); then applying a magnetic field ( 4 ) to the sample and irradiating the sample with a pulse sequence ( 5 ) that preferentially demagnetizes protons of the macromolecule ( 1,3 ) relative to the second proton ( 2 ); allowing the second proton ( 2 ) to exchange ( 6 ) with an exposed proton ( 1 ) of the macromolecule; and detecting the magnetization from the relatively magnetized second proton ( 2 ), which is now bound to the exposed position ( 1 c ) of the macromolecule. The invention also provides a method for observing a position in the macromolecule that bind a ligand.

1 . A method for preferentially observing an exposed position of a macromolecule,  
     comprising the steps of 
 (a) obtaining a sample comprising a macromolecule and a second molecule, wherein the macromolecule is larger than 35 kiloDaltons and has a position that is exposed to the second molecule,  
 wherein a first proton is bound to the exposed position of the macromolecule, a second proton is bound to the second molecule, and the first proton can exchange with the second proton;  
 (b) applying a magnetic field to the sample, thereby magnetizing the first proton and the second proton;  
 (c) irradiating the sample with a pulse sequence that preferentially demagnetizes the protons of the macromolecule relative to the second proton;  
 (d) allowing the second proton to exchange with the first proton, whereby the relatively magnetized second proton becomes bound to the exposed position of the macromolecule; and  
 (e) detecting the magnetization from the second proton;  
 whereby the exposed position of the macromolecule is preferentially observed.  
 
   
   
       2 . The method of  claim 1 , wherein the macromolecule is a polypeptide.  
   
   
       3 . The method of  claim 1 , wherein the macromolecule is larger than about 50 kDa.  
   
   
       4 . The method of  claim 1 , wherein the macromolecule is larger than about 75 kDa.  
   
   
       5 . The method of  claim 1 , wherein the macromolecule is larger than about 100 kDa.  
   
   
       6 . The method of  claim 1 , wherein the structure of the polypeptide has not been fully determined by an NMR technique.  
   
   
       7 . The method of  claim 1 , wherein resonances for fewer than 5% of the amino acids of the protein have been assigned by NMR techniques.  
   
   
       8 . The method of  claim 1 , wherein resonances for fewer than 10% of the amino acids of the protein have been assigned by NMR techniques.  
   
   
       9 . The method of  claim 1 , wherein resonances for fewer than 50% of the amino acids of the protein have been assigned by NMR techniques.  
   
   
       10 . The method of  claim 1 , wherein resonances for fewer than 75% of the amino acids of the protein have been assigned by NMR techniques.  
   
   
       11 . The method of  claim 1 , wherein the second molecule is a protic solvent.  
   
   
       12 . The method of  claim 1 , wherein the second molecule is water.  
   
   
       13 . The method of  claim 1 , wherein the position on the macromolecule that is exposed to the second molecule comprises 15N.  
   
   
       14 . The method of  claim 13 , wherein the pulse sequence comprises an  15 N filter.  
   
   
       15 . The method of  claim 1 , wherein the pulse sequence comprises the SEA pulse sequence.  
   
   
       16 . The method of  claim 1 , wherein step (c) further comprises  15 N,  1 H TROSY.  
   
   
       17 . The method of  claim 1 , wherein the pulse sequence comprises the SEA-TROSY pulse sequence.  
   
   
       18 . The method of  claim 1 , wherein step (d) occurs during a predetermined mixing time.  
   
   
       19 . The method of  claim 18 , wherein the mixing time is between 25 and 300 ms.  
   
   
       20 . The method of  claim 18 , wherein the mixing time is between 50 and 150 ms.  
   
   
       21 - 49 . (canceled)