Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 6, Pages 2538-2543Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.97.6.2538
Keywords
-
Categories
Funding
- NHLBI NIH HHS [R01 HL063203, HL63203] Funding Source: Medline
- NIGMS NIH HHS [P01 GM58890, P01 GM058890] Funding Source: Medline
Ask authors/readers for more resources
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 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 analysis of the H-1 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 [H-1, H-1]-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 H-1 and N-15 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 N-15/C-13 resonances upon nonselective labeling.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available