title: Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [beta (15N-valine)].
abstract: We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have (15)N-labeled the beta-chain Val residues and detected 16 of the 17 (1)H-(15)N 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 analysis of the (1)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 the (1)H and (15)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 backbone (15)N/(13)C resonances upon nonselective labeling.
authors.affiliation: Department of Chemistry, Structural Biology NMR Facility, Northwestern University, 2145 North Sheridan Road, Evanston, IL 60208, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Nocek
authors.forename: K
authors.initials: K
authors.lastname: Huang
authors.forename: B M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D000495:Allosteric Site; D002245:Carbon Dioxide / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014633:Valine / Q000737:chemistry
chemical_list: D006454:Hemoglobins; D002245:Carbon Dioxide; D014633:Valine