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.
- SourceAvailable from: Mary A Steinhardt[show abstract] [hide abstract]
ABSTRACT: Adverse drug event (ADE) reporting by pharmacists is an indispensable part of the drug safety system. U.S. pharmacists may submit reports of serious ADEs that they encounter to the Food and Drug Administration (FDA) through MedWatch. However, underreporting of serious ADEs is a common problem. Little is known about pharmacists' decision making with respect to ADE reporting. This study explored the utility of the theory of planned behavior (TPB) model in predicting Texas pharmacists' intention to report serious ADEs to the FDA. Data were collected from practicing Texas pharmacists using a mail questionnaire. A total of 1500 surveys were mailed, and 377 usable responses were obtained for a response rate of 26.4%. A majority (70.2%) of the 377 respondents were white/Caucasian, and 52.9% were male. Overall, pharmacists intended to report serious ADEs (mean=15.87±4.22; possible range: 3-21), had a positive attitude toward reporting (mean=4.62±4.92; possible range: -15 to +15), perceived that important others wanted them to report (subjective norm [SN] score=5.65±2.99; possible range: -9 to +9), and believed that they had control over their reporting behavior (perceived behavioral control [PBC] score=3.54±2.69; possible/actual range: -6 to +6). Attitude (β=0.221, P<.001) and SN (β=0.438, P<.001) significantly predicted intent; however, PBC (β=0.028, P>.05) did not. Attitude, SN, and PBC together accounted for 34.0% of the variance in intention to report serious ADEs (P<.001). The addition of past reporting behavior (P=.021) and perceived moral obligation (P<.001) significantly increased the variance in intention explained by the TPB model. Pharmacists showed a strong positive intent to report serious ADEs to the FDA. Strategies to increase pharmacists' intentions to report serious ADEs should focus on helping them see the value of reporting and altering their perception of social pressure toward reporting. The TPB may have utility in predicting ADE-reporting behavior.Research in Social and Administrative Pharmacy 11/2010; 7(4):369-82. · 2.35 Impact Factor
- ChemBioChem 09/2010; 11(14):1993-6. · 3.74 Impact Factor
- Clinical Biochemistry - CLIN BIOCHEM. 01/2011; 44(13).