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1.  Multiple ligand-specific conformations of the β2-adrenergic receptor 
Nature Chemical Biology  2011;7(10):692-700.
Seven-transmembrane receptors (7TMRs), also called G protein–coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this ‘biased agonism’ remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β2-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.
doi:10.1038/nchembio.634
PMCID: PMC3404607  PMID: 21857662
2.  Mapping mechanical properties of organic thin films by force-modulation microscopy in aqueous media 
Summary
The mechanical properties of organic and biomolecular thin films on surfaces play an important role in a broad range of applications. Although force-modulation microscopy (FMM) is used to map the apparent elastic properties of such films with high lateral resolution in air, it has rarely been applied in aqueous media. In this letter we describe the use of FMM to map the apparent elastic properties of self-assembled monolayers and end-tethered protein thin films in aqueous media. Furthermore, we describe a simple analysis of the contact mechanics that enables the selection of FMM imaging parameters and thus yields a reliable interpretation of the FMM image contrast.
doi:10.3762/bjnano.3.53
PMCID: PMC3458590  PMID: 23019540
acoustic atomic force microscopy; biomolecules; elastic modulus mapping; nanomechanical characterization; self-assembled monolayers
3.  A Miniaturized Technique for Assessing Protein Thermodynamics and Function Using Fast Determination of Quantitative Cysteine Reactivity 
Proteins  2011;79(4):1034-1047.
Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity (fQCR), which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.
doi:10.1002/prot.22932
PMCID: PMC3139720  PMID: 21387407
quantitative cysteine reactivity; thiol reactivity; protein thermodynamic stability; conformational free energy; protein folding kinetics; linkage analysis of protein stability; dissociation constants binding affinity; Staphylococcal nuclease; ribose-binding protein
4.  Probing the Folding Intermediate of Bacillus subtilis RNase P protein by NMR 
Biochemistry  2010;49(44):9428-9437.
Protein folding intermediates are often imperative to overall folding processes and consequent biological functions. However, the low population and transient nature of the intermediate states often hinder the biochemical and biophysical characterization. Previous studies have demonstrated that Bacillus subtilis ribonuclease P protein (P protein) is conformationally heterogeneous and folds with multiphasic kinetics, indicating the presence of an equilibrium and kinetic intermediate in its folding mechanism. In this study, NMR spectroscopy was used to study the ensemble corresponding to this intermediate (I). The results indicate that the N-terminal and C-terminal helical regions are mostly unfolded in I.1H-15N HSQC NMR spectra collected as a function of pH suggest that the protonation of His 22 may play a major role in the energetics of the equilibria between the unfolded, intermediate, and folded state ensembles of P protein. NMR paramagnetic relaxation enhancement experiments were also used to locate the small anion binding sites in both the intermediate and folded ensembles. The results for the folded protein are consistent with the previously modeled binding regions. These structural insights suggest a possible role for I in the RNase P holoenzyme assembly process.
doi:10.1021/bi100287y
PMCID: PMC3081390  PMID: 20843005
RNase P protein; NMR; Folding intermediate
5.  Osmolyte-Induced Folding of an Intrinsically Disordered Protein: Folding Mechanism in the Absence of Ligand 
Biochemistry  2010;49(25):5086-5096.
Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in the present study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine-N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations showed multiphasic kinetics. Equilibrium “cotitration” experiments were performed using both TMAO and urea during the titration to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are relatively slow compared to intrinsically folded proteins of similar size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.
doi:10.1021/bi100222h
PMCID: PMC2937257  PMID: 20476778
Protein folding; Osmolyte; Intrinsically disordered; RNase P protein

Results 1-5 (5)