Open AccessExtension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [β( 15 N-valine)]
Author(s) -
Judith M. Nocek,
KaiYao Huang,
Brian M. Hoffman
Publication year - 2000
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.97.6.2538
Subject(s) - chemistry , nuclear overhauser effect , nuclear magnetic resonance spectroscopy , allosteric regulation , relaxation (psychology) , context (archaeology) , tetramer , two dimensional nuclear magnetic resonance spectroscopy , crystallography , nuclear magnetic resonance , spectroscopy , chemical shift , valine , stereochemistry , amino acid , biochemistry , psychology , social psychology , paleontology , physics , quantum mechanics , biology , enzyme
We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each β-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have15 N-labeled the β-chain Val residues and detected 16 of the 171 H-15 N correlation peaks for β-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on analysis of the1 H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form. When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional [1 H,1 H]-NOESY-TROSY experiment (NOESY, NOE spectroscopy). We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample. This change induces substantial increments in the1 H and15 N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb. More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone15 N/13 C resonances upon nonselective labeling.