Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by 19F and 1H 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: Fluorine-containing amino acids are valuable probes for the biophysical characterization of proteins. Current methods for 19F-labeled protein production involve time-consuming genetic manipulation, compromised expression systems and expensive reagents. We show that Escherichia coli BL21, the workhorse of protein production, can utilise fluoroindole for the biosynthesis of proteins containing 19F-tryptophan.Chemical Communications 10/2012; 48(86):10681-10683. · 6.38 Impact Factor
- Clinical Biochemistry - CLIN BIOCHEM. 01/2011; 44(13).
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ABSTRACT: While many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, 19F NMR and near UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a water-soluble protein, which is biosynthetically enriched with 3-fluorophenylalanine (3F-Phe). 19F NMR solvent isotope shifts, resulting from replacing H2O with D2O, and paramagnetic shifts arising from dissolved O2 are used to monitor changes in water accessibility and hydrophobicity of the protein interior, as the protein progresses from a native state to an unfolded state, along a heat denaturation pathway. In comparison to the native state, solvent isotope shifts reveal the decreased presence of water in the hydrophobic core while paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N,1H and methyl 13C,1H HSQC NMR spectra identify that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.Biochemistry 08/2013; · 3.38 Impact Factor