Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [beta(N-15-valine)]

We present the first steps in applying transverse relaxation-optimized spec troscopy (TROSY) techniques to the study of allosterism, Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have N-15-labeled the beta-chain Vat residues and detected 16 of the 17 H-1-N-15 correlation peaks for beta-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on ana lysis of the H-1 pseudocontact-shift increments produced by oxidizing the d iamagnetic R state HbCO to the paramagnetic R state fluoromet form. When po ssible, we support these assignments with sequential nuclear Overhauser eff ect (NOE) information obtained from a two-dimensional [H-1, H-1]-NOESY-TROS Y experiment (NOESY, NOE spectroscopy). We have induced further the R-T con formational change by adding the allosteric effector, inositol hexaphosphat e, to the fluoromet-Hb sample. This change induces substantial increments i n the H-1 and N-15 chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preli minary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring th e allosteric response of the ensemble-averaged solution structure of Hb. Mo re important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone N-15/C-13 resonances upon nonselectiv e labeling.