pubmed.ncbi.nlm.nih.gov

Tailoring relaxation dispersion experiments for fast-associating protein complexes - PubMed

  • ️Mon Jan 01 2007

. 2007 Nov 7;129(44):13406-7.

doi: 10.1021/ja0762238. Epub 2007 Oct 13.

Affiliations

Tailoring relaxation dispersion experiments for fast-associating protein complexes

Kenji Sugase et al. J Am Chem Soc. 2007.

Abstract

NMR relaxation dispersion spectroscopy is a powerful technique to elucidate the mechanism of protein-protein binding reactions. However, it is difficult to optimize the concentration ratios that give relaxation dispersions of appropriate amplitude to determine accurate kinetic and thermodynamic parameters, especially in cases of very tight binding. In this study, we have obtained 15N R2 dispersions of Asn803-hydroxylated hypoxia-inducible factor-1α (HIF-OH) in the presence of sub-stoichiometric amount of its target protein, the transcriptional adapter zinc-binding (TAZ1) domain of CREB binding protein, whereas no R2 dispersion was observed for the bound state of HIF-OH at 1:1 concentration ratio because the binding is too tight. Although the R2 dispersions were measured for the free peaks of HIF-OH, they enabled us to quantitate the kinetic and thermodynamic parameters of HIF-OH/TAZ1 binding process. Simulations of effective R2 rates revealed that the association rate is the key factor to determine the amplitude of R2 dispersions. By careful optimization of the concentration ratio, the R2 dispersion method should be generally applicable for studying a wide range of protein-protein, protein-nucleic acid, and protein-small molecule interactions.

PubMed Disclaimer

Figures

Figure 1
Figure 1

15N R2 dispersion profiles for Arg820 of HIF-OH recorded for 510 μM [15N]-HIF-OH in the presence of sub-stoichiometric amounts of TAZ1. Data were acquired at 900 MHz (filled circles) and 600 MHz (open circles) using relaxation compensated Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences., Curves for all observable dispersions are shown in Supplementary Figure S3.

Figure 2
Figure 2

R2eff rates for Arg820 of HIF-OH simulated using the parameters listed in Table 1. (a) R2eff rates versus the concentration ratio, [TAZ1]0/[HIF-OH]0. The red lines indicate R20, where the R20 rates for the free and bound states are 5 and 15 s−1, respectively. The grey area indicates the concentration ratios that give Rex rates from 3 to 25 s−1. A similar plot for the pKID/KIX system is shown in Figure S5. (b) R2 dispersion profiles at concentration ratios of 0.97, 0.98, 0.99 and 1. τCP indicates the delay between successive 15N 180 pulses in the CPMG sequence.

Similar articles

Cited by

References

    1. Loria JP, Rance M, Palmer AG. J Am Chem Soc. 1999;121:2331–2332.
    1. Mulder FA, Mittermaier A, Hon B, Dahlquist FW, Kay LE. Nat Struct Biol. 2001;8:932–935. - PubMed
    1. Boehr DD, McElheny D, Dyson HJ, Wright PE. Science. 2006;313:1638–1642. - PubMed
    1. McCammon JA, Harvey SC. Dynamics of Proteins and Nucleic Acids. Cambridge University Press; Cambridge: 1987.
    1. Korzhnev DM, Salvatella X, Vendruscolo M, Di Nardo AA, Davidson AR, Dobson CM, Kay LE. Nature. 2004;430:586–590. - PubMed

Publication types

MeSH terms

Substances