Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide.
A method for the measurement of proton T(1)(rho) relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90 degrees excitation in a HOHAHA pulse sequence. The experiment is applied to alpha-bungarotoxin (alpha-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) alpha-subunit. A good correlation between high T(1)(rho) values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the alpha-BTX complex with an AChR peptide and the model for alpha-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor alpha-subunit is exhibited, while residues implicated in binding gamma- and delta-subunits retain their mobility. In addition, the quantitative T(1)(rho) measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.
Samson
Abraham O
AO
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Chill
Jordan H
JH
Anglister
Jacob
J
D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry* / Q000378:metabolism; D009038:Motion; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011522:Protons; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000378:metabolism*
D002038:Bungarotoxins; D010446:Peptide Fragments; D011522:Protons; D011950:Receptors, Cholinergic