Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19 F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19 F spectra to those from two-dimensional 1 H-15 N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19 F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19 F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

Nearly all biological processes, including strictly regulated protein−protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen-activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19 F NMR lineshape analysis. We also analyze the temperature dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3−peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically relevant cosolutes. Although most of the protein cosolutes destabilize the SH3−peptide complexes, the effects are nongeneralizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein−protein interactions in cells and show the importance of using physiologically relevant cosolutes for investigating macromolecular crowding effects.

ID 16458 Poster Board 571Alcohol use disorder is a prevalent psychiatric disorder affecting nearly 15 million people in the United States. Individuals who wish to cease to consume alcohol are often met with the effects of the dark side of addiction after alcohol withdrawal. One detrimental withdrawal symptom is increased pain sensitivity, which may contribute to relapse to attain the analgesic effect of alcohol. The present study investigated alcohol withdrawal-induced allodynia in a mouse model of chronic ethanol administration and assessed the effectiveness of a histone deacetylase inhibitor in preventing the development of allodynia. Male and female C57BL/6J mice were first given Lieber-DeCarli control liquid diet for 5 days. The mice were then separated into control and ethanol (5% vol/ vol) diet groups and fed their respective diets for 10 days. On day 11, the ethanol and control diet-fed mice were given an oral gavage of either ethanol (5 g kg -1 body weight) or an isocaloric maltose/dextran solution, respectively. Ethanol diet-fed mice then underwent ethanol withdrawal for 24 hours. We found that mice given ethanol diet plus withdrawal exhibited allodynia compared to control mice. The allodynic response persisted for at least 48 hours and then slowly returned to baseline levels. Finally, we performed an experiment in which we treated mice during ethanol withdrawal with suberoyl anilide hydroxamic acid (SAHA), a panhistone deacetylase (HDAC) inhibitor and found that SAHA attenuated the allodynic response in ethanol diet-fed plus withdrawal mice. These results demonstrate that chronic ethanol diet plus withdrawal induces allodynia, and SAHA prevents the development of this pain. This model can be used to further understand the molecular mechanisms and the effectiveness of pharmacological interventions in ethanol withdrawal-induced pain sensitivity.

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