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

Judith M. Nocek, Kai Huang, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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 have 15N-labeled the β-chain Val residues and detected 16 of the 17 1H-15N 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 the 1H 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 [1H,1H]-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 1H and 15N 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 15N/13C resonances upon nonselective labeling.

Original languageEnglish (US)
Pages (from-to)2538-2543
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number6
DOIs
StatePublished - Mar 14 2000

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [β(<sup>15</sup>N-valine)]'. Together they form a unique fingerprint.

Cite this