TY - JOUR

T1 - Slow dynamics in folded and unfolded states of an SH3 domain

AU - Tollinger, M

AU - Skrynnikov, NR

AU - Mulder, FAA

AU - Forman-Kay, JD

AU - Kay, LE

PY - 2001/11/21

Y1 - 2001/11/21

N2 - N-15 relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F-exch) and unfolded (U-exch) states under nondenaturing conditions in a ratio of 2:1 at 20 degreesC, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U-exch ensemble there is a contiguous stretch of residues undergoing conformational exchange on a mus/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F-exch <----> U-exch conformational. exchange process and the mus/ms exchange event within the U-exch state contribute to the 15N line width and can be analyzed using CPMG-based 15N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental 15N relaxation dispersion data with results from an 15N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.

AB - N-15 relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F-exch) and unfolded (U-exch) states under nondenaturing conditions in a ratio of 2:1 at 20 degreesC, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U-exch ensemble there is a contiguous stretch of residues undergoing conformational exchange on a mus/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F-exch <----> U-exch conformational. exchange process and the mus/ms exchange event within the U-exch state contribute to the 15N line width and can be analyzed using CPMG-based 15N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental 15N relaxation dispersion data with results from an 15N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.

KW - NUCLEAR-MAGNETIC-RESONANCE

KW - TIME-SCALE DYNAMICS

KW - N-15 NMR RELAXATION

KW - CHEMICAL-EXCHANGE

KW - BACKBONE DYNAMICS

KW - STRUCTURAL-CHARACTERIZATION

KW - STAPHYLOCOCCAL NUCLEASE

KW - HETERONUCLEAR NMR

KW - PROTEIN DYNAMICS

KW - DENATURED STATES

U2 - 10.1021/ja011300z

DO - 10.1021/ja011300z

M3 - статья

VL - 123

SP - 11341

EP - 11352

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 46

ER -