Tryptophan Solvent Exposure in Folded and Unfolded States of an SH3 Domain by 19F and 1H NMR

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• 作者：Ferenc Evanics ; Irina Bezsonova ; Joseph Marsh ; Julianne L. Kitevski ; Julie D. Forman-Kay ; R. Scott Prosser
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：November 28, 2006
• 年：2006
• 卷：45
• 期：47
• 页码：14120 - 14128
• 全文大小：175K
• 年卷期：v.45,no.47(November 28, 2006)
• ISSN：1520-4995

文摘

The isolated N-terminal SH3 domain of the Drosophila signal transduction protein Drk (drkNSH3) is a useful model for the study of residual structure and fluctuating structure in disordered proteinssince it exists in slow exchange between a folded (F_exch) and compact unfolded (U_exch) state in roughlyequal proportions under nondenaturing conditions. The single tryptophan residue, Trp36, is believed toplay a key role in forming a non-native hydrophobic cluster in the U_exch state, with a number of long-range nuclear Overhauser contacts (NOEs) observed primarily to the indole proton. Substitution of Trp36for 5-fluoro-Trp36 resulted in a substantial shift in the equilibrium to favor the F_exch state. A variety of¹⁹F NMR measurements were performed to investigate the degree of solvent exposure and hydrophobicityassociated with the 5-fluoro position in both the F_exch and U_exch states. Ambient T₁ measurements andH₂O/D₂O solvent isotope effects indicated extensive protein contacts to the 5-fluoro position in the F_exchstate and greater solvent exposure in the U_exch state. This was corroborated by the measurements ofparamagnetic effects (chemical shift perturbations and T₁ relaxation enhancement) from dissolved oxygenat a partial pressure of 20 atm. In contrast, paramagnetic effects from dissolved oxygen revealed lesssolvent exposure to the indole proton of Trp36 in the U_exch state than that observed for the F_exch state,consistent with the model in which Trp36 indole belongs to a non-native cluster. Thus, although the U_exchstate may be described as a dynamically interconverting ensemble of conformers, there appears to besignificant asymmetry in the environment of the indole group and the six-membered ring or backbone ofTrp36. This implied lack of averaging of a side chain position is in contrast to the general view of fluctuatingside chains within disordered states.