Tryptophan Solvent Exposure in Folded and Unfolded States of an SH3 Domain by 19 F and 1 H NMR †
Department of Chemistry, University of Toronto, Toronto, Ontario, CanadaBiochemistry (Impact Factor: 3.02). 12/2006; 45(47):14120-8. DOI: 10.1021/bi061389r
The isolated N-terminal SH3 domain of the Drosophila signal transduction protein Drk (drkN SH3) is a useful model for the study of residual structure and fluctuating structure in disordered proteins since it exists in slow exchange between a folded (Fexch) and compact unfolded (Uexch) state in roughly equal proportions under nondenaturing conditions. The single tryptophan residue, Trp36, is believed to play a key role in forming a non-native hydrophobic cluster in the Uexch state, with a number of long-range nuclear Overhauser contacts (NOEs) observed primarily to the indole proton. Substitution of Trp36 for 5-fluoro-Trp36 resulted in a substantial shift in the equilibrium to favor the Fexch state. A variety of 19F NMR measurements were performed to investigate the degree of solvent exposure and hydrophobicity associated with the 5-fluoro position in both the Fexch and Uexch states. Ambient T1 measurements and H2O/D2O solvent isotope effects indicated extensive protein contacts to the 5-fluoro position in the Fexch state and greater solvent exposure in the Uexch state. This was corroborated by the measurements of paramagnetic effects (chemical shift perturbations and T1 relaxation enhancement) from dissolved oxygen at a partial pressure of 20 atm. In contrast, paramagnetic effects from dissolved oxygen revealed less solvent exposure to the indole proton of Trp36 in the Uexch state than that observed for the Fexch state, consistent with the model in which Trp36 indole belongs to a non-native cluster. Thus, although the Uexch state may be described as a dynamically interconverting ensemble of conformers, there appears to be significant asymmetry in the environment of the indole group and the six-membered ring or backbone of Trp36. This implied lack of averaging of a side chain position is in contrast to the general view of fluctuating side chains within disordered states.
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ABSTRACT: Due to their dynamic ensemble nature and a deficiency of experimental restraints, disordered states of proteins are difficult to characterize structurally. Here, we have expanded upon our previous work on the unfolded state of the Drosophila drk N-terminal (drkN) SH3 domain with our program ENSEMBLE, which assigns population weights to pregenerated conformers in order to calculate ensembles of structures whose properties are collectively consistent with experimental measurements. The experimental restraint set has been enlarged with newly measured paramagnetic relaxation enhancements from Cu(2+) bound to an amino terminal Cu(2+)-Ni(2+) binding (ATCUN) motif as well as nuclear Overhauser effect (NOE) and hydrogen exchange data from recent studies. In addition, two new pseudo-energy minimization algorithms have been implemented that have dramatically improved the speed of ENSEMBLE population weight assignment. Finally, we have greatly improved our conformational sampling by utilizing a variety of techniques to generate both random structures and structures that are biased to contain elements of native-like or non-native structure. Although it is not possible to uniquely define a representative structural ensemble, we have been able to assess various properties of the drkN SH3 domain unfolded state by performing ENSEMBLE minimizations of different conformer pools. Specifically, we have found that the experimental restraint set enforces a compact structural distribution that is not consistent with an overall native-like topology but shows preference for local non-native structure in the regions corresponding to the diverging turn and the beta5 strand of the folded state and for local native-like structure in the region corresponding to the beta6 and beta7 strands. We suggest that this approach could be generally useful for the structural characterization of disordered states.Journal of Molecular Biology 05/2007; 367(5):1494-510. DOI:10.1016/j.jmb.2007.01.038 · 4.33 Impact Factor
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ABSTRACT: AbstractIntermolecular Overhauser effects resulting from interactions of solvent molecules with a solute are readily detected with modern instrumentation. After a discussion of how these effects arise, applications of intermolecular Overhauser effects in studies of solute–solvent and other interactions in solution are reviewed. Observed effects may reflect (a) a local concentration of solvent molecules near the solute that is different from the concentration of the bulk, (b) kinematic behavior of solvent molecules near the solute that is different from that characteristic of the bulk solvent, or (c) formation of solute–solvent complexes that are long-lived relative to the time associated with diffusive encounters of solute and solvent. In mixed solvents, a solute–solvent Overhauser effect may indicate preferential association of one solvent component with the solute; such interactions may be regiospecific. Determination of solvent–solute intermolecular Overhauser effects have been done in systems that range from solutions of monoatomic gases to proteins. It is shown that additional experimental developments are needed to enable unambiguous interpretation of observed effects.Annual Reports on NMR Spectroscopy 01/2008; 64(64):21-215. DOI:10.1016/S0066-4103(08)00002-1 · 2.27 Impact Factor
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ABSTRACT: This review focuses on the applications of dissolved oxygen in NMR studies of protein topology. A brief discussion is given to explain the origin of O2-induced paramagnetic shifts and relaxation rate enhancements, which are seen for a variety of nuclei of biological interest—in particular 13C, 19F, and 1H. We also give examples of applications of paramagnetic effects from dissolved O2, which include studies of solvent exposure, hydrophobicity, transient contacts or local clustering in intrinsically disordered proteins, immersion depth in membranous systems, and topology of membrane proteins. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 239–253, 2008.Concepts in Magnetic Resonance Part A 07/2008; 32A(4):239 - 253. DOI:10.1002/cmr.a.20118 · 1.00 Impact Factor
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