Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by F-19 and H-1 NMR
ABSTRACT 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: Disordered states of proteins include the biologically functional intrinsically disordered proteins and the unfolded states of normally folded proteins. In recent years, ensemble-modeling strategies using various experimental measurements as restraints have emerged as powerful means for structurally characterizing disordered states. However, these methods are still in their infancy compared with the structural determination of folded proteins. Here, we have addressed several issues important to ensemble modeling using our ENSEMBLE methodology. First, we assessed how calculating ensembles containing different numbers of conformers affects their structural properties. We find that larger ensembles have very similar properties to smaller ensembles fit to the same experimental restraints, thus allowing a considerable speed improvement in our calculations. In addition, we analyzed the contributions of different experimental restraints to the structural properties of calculated ensembles, enabling us to make recommendations about the experimental measurements that should be made for optimal ensemble modeling. The effects of different restraints, most significantly from chemical shifts, paramagnetic relaxation enhancements and small-angle X-ray scattering, but also from other data, underscore the importance of utilizing multiple sources of experimental data. Finally, we validate our ENSEMBLE methodology using both cross-validation and synthetic experimental restraints calculated from simulated ensembles. Our results suggest that secondary structure and molecular size distribution can generally be modeled very accurately, whereas the accuracy of calculated tertiary structure is dependent on the number of distance restraints used. Proteins 2011. © 2011 Wiley Periodicals, Inc.Proteins Structure Function and Bioinformatics 02/2012; 80(2). DOI:10.1002/prot.23220 · 2.92 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
- Clinical Biochemistry 09/2011; 44(13). DOI:10.1016/j.clinbiochem.2011.08.672 · 2.23 Impact Factor