(15)N 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 degrees C, 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 micros/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 micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N 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.
Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
Although intrinsically disordered proteins (IDPs) are widespread in nature and play diverse and important roles in biology, they have to date been little characterized structurally. Auspiciously, intensified efforts using NMR spectroscopy have started to uncover the breadth of their conformational landscape. In particular, polypeptide backbone chemical shifts are emerging as powerful descriptors of local dynamic deviations from the "random coil" state toward canonical types of secondary structure. These digressions, in turn, can be connected to functional or dysfunctional protein states, for example, in adaptive molecular recognition and protein aggregation. Here we describe a first inventory of IDP backbone (15)N, (1)H(N), (1)H(α), (13)C(O), (13)C(β), and (13)C(α) chemical shifts using data obtained for a set of 14 proteins of unrelated sequence and function. Singular value decomposition was used to parametrize this database of 6903 measured shifts collectively in terms of 20 amino acid-specific random coil chemical shifts and 40 sequence-dependent left- and right-neighbor correction factors, affording the ncIDP library. For natively unfolded proteins, random coil backbone chemical shifts computed from the primary sequence displayed root-mean-square deviations of 0.65, 0.14, 0.12, 0.50, 0.36, and 0.41 ppm from the experimentally measured values for the (15)N, (1)H(N), (1)H(α), (13)C(O), (13)C(β), and (13)C(α) chemical shifts, respectively. The ncIDP prediction accuracy is significantly higher than that obtained with libraries for small peptides or "coil" regions of folded proteins.
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