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
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ABSTRACT: Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild type protein. We created a test cPYP by connecting the N- and C- termini of wild type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C- termini at G115 and S114 respectively. Biophysical analysis indicated that this cPYP adopts a dark state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark state cPYP is ~10-fold faster than wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ~10 min. NMR, circular dichroism, and UV-Vis analysis indicated that the M121E-cPYP mutant also adopts a dark state structure like that of wtPYP although protonated and deprotonated forms of the chromophore coexist giving rise to a shoulder near 380 nm in the UV-Vis absorption spectrum. Fluorine NMR studies with fluoro-tryptophan labeled M121E-cPYP show that blue-light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering) consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs by replacing the linker polypeptide with a target protein or peptide sequence.Biochemistry 04/2013; · 3.38 Impact Factor
- Clinical Biochemistry - CLIN BIOCHEM. 01/2011; 44(13).