Terrence G. Oas

Conformational selection or induced fit: A flux description of reaction mechanism

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the “induced fit” mechanism (binding first) or “conformational selection” (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.

Rotary resonance recoupling of dipolar interactions in solid‐state nuclear magnetic resonance spectroscopy

A new resonance effect in solid‐state nuclear magnetic resonance (NMR) is described. The effect involves a combination of magic‐angle sample rotation with irradiation of a heteronuclear spin system at the Larmor frequency of one of the spin species. If the irradiation intensity is such as to establish a match between spin nutation and sample rotation, it is shown that the heteronuclear dipolar spin interaction is selectively reintroduced into the spectrum. This allows small dipolar coupling constants to be measured in the presence of large shielding anisotropies. Applications are anticipated for determination of internuclear distances in materials lacking long‐range order, such as polycrystalline materials, polymers, and surfaces.

Quantitative protein stability measurement in vivo

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Eschericia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric λ repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.

The Active Conformation of β-Arrestin1 DIRECT EVIDENCE FOR THE PHOSPHATE SENSOR IN THE N-DOMAIN AND CONFORMATIONAL DIFFERENCES IN THE ACTIVE STATES OF β-ARRESTINS1 AND -2

β-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that β-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and β-arrestin2, these studies are not representative of conformational changes in β-arrestin1. Accordingly, in this study, we determine conformational changes in β-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V2 vasopressin receptor (V2Rpp) or the corresponding unphosphorylated peptide (V2Rnp). V2Rpp binds specifically to β-arrestin1 causing significant conformational changes, whereas V2Rnp does not alter the conformation of β-arrestin1. Upon V2Rpp binding, we show that the previously shielded Arg393 becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg285 becomes more accessible, and this residue is located in a region of β-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate “activation” of β-arrestin1, and we also show a functional consequence of the release of the C terminus of β-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of β-arrestin1 in the presence of V2Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in β-arrestin1 when compared with β-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the “receptor-bound” conformations of β-arrestins1 and 2 are different.

Fast and faster: A designed variant of the B‐domain of protein A folds in 3 μsec

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B‐domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 μsec. Although wild‐type BdpA folds extremely fast, simple‐point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all‐atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence‐based time‐resolved temperature‐jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity

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, 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. Proteins 2011.

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

The Gibbs free energy difference between native and unfolded states (“stability”) is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Methionine oxidation of monomeric λ repressor : The denatured state ensemble under nondenaturing conditions

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric λ repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of α‐helical structure in these conformations is detected by far‐UV circular dichroism, and some tertiary interactions are suggested by near‐UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

Enhancement of the effect of small anisotropies in magic-angle spinning nuclear magnetic resonance

A variety of novel methods in magic-angle spinning NMR is described. The effects have in common the enhancement of the influence of small anisotropies on the NMR spectrum by deliberate intervention, i.e. either by applying pulses, carefully chosen continuous r.f. fields, or by adjustment of the spinning speed. It is shown that rotational sidebands in two-dimensional spin-echo NMR are often much larger than in one-dimensional NMR, owing to the interference of the π-pulse with the rotational echo formation. It is also demonstrated that in systems containing heteronuclear spin pairs the application of a weak continuous r.f. field of carefully chosen intensity can reintroduce small heteronuclear couplings into the spectrum. This is known as rotational resonance recoupling and is due to the interference of coherent spin rotations with the normal averaging effect of the sample rotation. Related effects can occur in homonuclear spin systems when an integer multiple of the spinning speed matches the difference between isotropic chemical shifts. In this case no extra r.f. field is necessary to amplify the effect of the non-secular parts of the dipolar interaction. Greatly enhanced polarization exchange as well as strong spectral effects are demonstrated. The application of these novel methods to the measurement of small interaction tensors and thereby to the extraction of important structural information is discussed.

Suppression of conformational heterogeneity at a protein-protein interface.

Significance The emergence of antibiotic-resistant strains of bacteria, such as methicillin-resistant Staphylococcus aureus, is an increasing threat to human health. S. aureus infections cause a variety of health complications, ranging from skin lesions to life-threatening infections. Staphylococcal protein A (SpA) is the major cell-surface protein and a multitarget virulence factor. The design of SpA-targeted therapeutics requires a molecular description of its interactions with host proteins. Here we report the crystal structure of a complete SpA domain in complex with an Fc fragment of human IgG. Our structure reveals changes in SpA when it binds to Fc, including a significant reduction in conformational heterogeneity as well as displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium’s evasion of the host immune system. SpA includes five small three-helix–bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein–protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.