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A Joshua Wand

Publications and source records attributed to A Joshua Wand.

At least 19 recordsLinked to original sources
PubMed2006

Revealing the nature of the native state ensemble through cold denaturation.

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.

Animals↗
PubMed2006

Cold denaturation of encapsulated ubiquitin.

Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multistate cold-induced unfolding of the protein.

Cold Temperature↗
PubMed2006

Characterization of the backbone and side chain dynamics of the CaM-CaMKIp complex reveals microscopic contributions to protein conformational entropy.

Calmodulin is a central mediator of calcium-dependent signal transduction pathways and regulates the activity of a large number of diverse targets. Calcium-dependent interactions of calmodulin with regulated proteins are of generally high affinity but of quite variable thermodynamic origins. Here we investigate the influence of the binding of the calmodulin-binding domain of calmodulin kinase I on the fast internal dynamics of calcium-saturated calmodulin. NMR relaxation was used to probe motion on the backbone (viewed through the backbone amide NH group) and the side chains (viewed through methyl groups). The distribution of the amplitudes of side chain dynamics is trimodal. The microscopic details of side chain motion are compared with those of a thermodynamically and structurally similar complex of calmodulin with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. While there are no significant differences in backbone dynamics and no net change in methyl-bearing side chain dynamics, a large redistribution of the amplitude of methyl dynamics is observed between the two complexes. The variation in dynamics was largely localized to the heterogeneously dynamic target-binding interface, suggesting that differential dynamics of the binding surface plays a functional role in the high-affinity binding interactions of calmodulin. These results begin to reveal a fundamental role for residual protein entropy in molecular recognition by calmodulin.

Amino Acid Sequence↗
PubMed2006

Conformational dynamics of calmodulin in complex with the calmodulin-dependent kinase kinase alpha calmodulin-binding domain.

As the primary intracellular calcium sensor, calcium-saturated calmodulin (CaM) regulates numerous and diverse proteins. Several mechanisms, including tissue-specific expression, localization, and sequestration, work in concert to limit the total number of available targets of calmodulin within a cell. While the free energies of binding of calmodulin-binding domains of regulated proteins by CaM have been shown to be highly similar, they result from vastly different enthalpic and entropic contributions. Here, we report the backbone and side-chain methyl dynamics of calcium-activated calmodulin in complex with a peptide corresponding to the CaM-binding domain of calmodulin kinase kinase, along with the thermodynamic underpinnings of complex formation. The results show a considerable reduction in side-chain mobility throughout CaM upon binding the CaMKKalpha peptide, which is consistent with the enthalpically driven nature of the binding. Site-specific comparison to another kinase-derived peptide complex with similar thermodynamic values reveals significant differences in dynamics largely localized to the hydrophobic binding sites.

Animals↗
PubMed2006

Arylamide derivatives as peptidomimetic inhibitors of calmodulin.

[structure: see text] Many peptides bind to calmodulin (CaM) in a helical conformation. Here we describe a group of synthetic inhibitors of CaM based on an arylamide scaffold that is intended to mimic smMLCK, a CaM-binding helical peptide. Compound 1 showed a K(i) value of 7.10 +/- 1.48 nM in a fluorescence polarization assay that monitors the strong association of CaM and its peptide ligand mastoparan X. ((1)H,(15)N)-HSQC NMR spectroscopy experiments suggested that 1 binds to CaM in an analogous fashion to that of smMLCK.

Amides↗
PubMed2005

New reverse micelle surfactant systems optimized for high-resolution NMR spectroscopy of encapsulated proteins.

Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) is a surfactant commonly used to encapsulate water soluble proteins within the aqueous core of a reverse micelle. In the context of high-resolution NMR studies of encapsulated proteins the size of the resulting reverse micelle is critically important. We have designed and synthesized a short AOT analogue, 3,3-dimethyl-1-butylsulfosuccinate sodium salt and determined that it is able to form reverse micelles and to encapsulate the protein ubiquitin with high structural fidelity. AOT is often found to significantly destabilize encapsulated proteins, largely through charge-charge interactions between the anionic headgroup and the surface of the protein. Here we demonstrate, for the first time, that proportional mixtures of anionic and cationic surfactants can form reverse micelles that are also capable of protein encapsulation with high fidelity.

Cations↗
PubMed2005

Novel surfactant mixtures for NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids.

NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids is emerging as a tool for biophysical studies of proteins in atomic detail in a variety of otherwise inaccessible contexts. The central element of the approach is the encapsulation of the protein of interest within the aqueous core of a reverse micelle with high structural fidelity. The process of encapsulation is highly dependent upon the nature of the surfactant(s) employed. Here we describe novel mixtures of surfactants that are capable of successfully encapsulating a range of types of proteins under a variety of conditions.

Cetrimonium↗
PubMed2005

Backbone and side chain dynamics of mutant calmodulin-peptide complexes.

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.

Allosteric Site↗
PubMed2005

High-resolution NMR studies of encapsulated proteins in liquid ethane.

Many of the difficulties presented by large, aggregation-prone, and membrane proteins to modern solution NMR spectroscopy can be alleviated by actively seeking to increase the effective rate of molecular reorientation. An emerging approach involves encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid, such as the short chain alkanes. Here we present the encapsulation of proteins with high structural fidelity within reverse micelles dissolved in liquid ethane. The addition of appropriate cosurfactants can significantly reduce the pressure required for successful encapsulation. At these reduced pressures, the viscosity of the ethane solution is low enough to provide sufficiently rapid molecular reorientation to significantly lengthen the spin-spin NMR relaxation times of the encapsulated protein.

Cytochromes c↗
PubMed2005

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin.

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.

Carbon Isotopes↗
PubMed2005

NMR spectroscopy of proteins encapsulated in a positively charged surfactant.

Traditionally, large proteins, aggregation-prone proteins, and membrane proteins have been difficult to examine by modern multinuclear and multidimensional solution NMR spectroscopy. A major limitation presented by these protein systems is that their slow molecular reorientation compromises many aspects of the more powerful solution NMR methods. Several approaches have emerged to deal with the various spectroscopic difficulties arising from slow molecular reorientation. One of these takes the approach of actively seeking to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid. Since the encapsulation is largely driven by electrostatic interactions, the preparation of samples of acidic proteins suitable for NMR spectroscopy has been problematic owing to the paucity of suitable cationic surfactants. Here, it is shown that the cationic surfactant CTAB may be used to prepare samples of encapsulated anionic proteins dissolved in low viscosity solvents. In a more subtle application, it is further shown that this surfactant can be employed to encapsulate a highly basic protein, which is completely denatured upon encapsulation using an anionic surfactant.

Animals↗
PubMed2005

Changes in calmodulin main-chain dynamics upon ligand binding revealed by cross-correlated NMR relaxation measurements.

The fast dynamics of protein backbones are often investigated by nuclear magnetic relaxation experiments that report on the degree of spatial restriction of the amide bond vector. By comparing calmodulin in the peptide-bound and peptide-free states with these classical methods, we observe little difference in the dynamics of the polypeptide main chain (average order parameter decrease of 0.01 unit upon binding). However, when using NMR methods that monitor the mobility of the CO-Calpha bond vector, we reveal a significant reduction of dynamics of the protein main chain (average order parameter decrease of 0.048 units). Previous investigations have suggested that the side-chain dynamics is reduced by an average of 0.07 order parameter units upon ligand binding (Lee, A. L.; Kinnear, S. A.; Wand, A. J. Nat. Struct. Biol. 2000, 7, 72-77). The current findings suggest that the change of the CO-Calpha bond vector dynamics is intermediate between the changes in NH and side-chain dynamics and report a previously undetected loss of main-chain entropy. Weak site-to-site correlations between the different motional indicators are also observed.

Binding Sites↗
PubMed2005

Self contained high pressure cell, apparatus and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for NMR spectroscopy.

The design of a sample cell for high performance nuclear magnetic resonance (NMR) at elevated pressure is described. The cell has been optimized for the study of encapsulated proteins dissolved in low viscosity fluids but is suitable for more general NMR spectroscopy of biomolecules at elevated pressure. The NMR cell is comprised of an alumina toughened zirconia tube mounted on a self-sealing non-magnetic metallic valve. The cell has several advantages including relatively low cost, excellent NMR performance, high pressure tolerance, chemical inertness and a relatively large active volume. Also described is a low volume sample preparation device which allows for the preparation of samples under high hydrostatic pressure and their subsequent transfer to the NMR cell.

Journal Article↗
PubMed2004

Forced folding and structural analysis of metastable proteins.

A significant fraction of the proteins encoded by the human and other genomes appears to be significantly unfolded in vitro. This will undoubtedly hamper attempts to characterize their structure by classical crystallographic or solution NMR methods. Here we show that encapsulation of a metastable protein within the restricted volume a reverse micelle can be used to force fold the protein and allow its characterization by modern methods of NMR spectroscopy. This may have significant utility in the context of structural proteomics. In addition, variation of the inner volume of the reverse micelle can be used to probe the character of the manifold of unfolded states.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed2004

De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1.

An idealized, water-soluble D(2)-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(1). Each heme is coordinated by two His residues from diagonally apposed helices. In the model, the imidazole rings of the His ligands are held in a somewhat unusual perpendicular orientation as found in cytochrome bc(1), which is maintained by a second-shell hydrogen bond to a Thr side chain on a neighboring helix. The resulting peptide is unfolded in the apo state but assembles cooperatively upon binding to heme into a well-folded tetramer. Each tetramer binds two hemes with high affinity at low micromolar concentrations. The equilibrium reduction midpoint potential varies between -76 mV and -124 mV vs SHE in the reducing and oxidizing direction, respectively. The EPR spectrum of the ferric complex indicates the presence of a low-spin species, with a g(max) value of 3.35 comparable to those obtained for hemes b of cytochrome bc(1) (3.79 and 3.44). This provides strong support for the designed perpendicular orientation of the imidazole ligands. Moreover, NMR spectra show that the protein exists in solution in a unique conformation and is amenable to structural studies. This protein may provide a useful scaffold for determining how second-shell ligands affect the redox potential of the heme cofactor.

Binding Sites↗
PubMed2004

Assignments of carbon NMR resonances for microcrystalline ubiquitin.

Solid-state NMR 2D spectroscopy was used to correlate carbon backbone and side-chain chemical shifts for uniformly (13)C,(15)N-enriched microcrystalline ubiquitin. High applied field strengths, 800 MHz for protons, moderate proton decoupling fields, 80-100 kHz, and high magic angle sample spinning frequencies, 20 kHz, were used to narrow the most of the carbon line widths to 0.5-0.8 ppm. Homonuclear magnetization transfer was effected by matching the proton RF field to the spinning frequency, the so-called dipolar-assisted rotational resonance (DARR) (Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett. 2001, 344, 631-637), and a mixing time of 20 ms was used to maximize the intensity of one-bond transfers between carbon atoms. This polarization transfer sequence resulted in roughly 14% transfer efficiencies for directly bonded carbon pairs and 4% transfer efficiencies for carbons separated by a third carbon. With this simple procedure, the majority of the one-bond correlations was observed with moderate transfer efficiencies, and many two-bond correlations were also observed with weaker intensities. Spin systems could be identified for more than half of the amino acid side chains, and site-specific assignments were readily possible via comparison with 400 MHz (15)N-(13)C-(13)C correlation spectroscopy (described separately).

Amino Acids↗
PubMed2004

Assignment of the backbone resonances for microcrystalline ubiquitin.

Site-specific assignments for the solid-state NMR spectra of uniformly 13C,15N-enriched ubiquitin are described. The assignments are derived from three three-dimensional 15N-13C-13C correlation spectra collected at 400 MHz on microcrystalline material. A few residues (the loop near Threonine 9 and the C-terminal fragment) were missing and correspond to regions previously reported to be mobile on the basis of X-ray crystallography and solution NMR studies. A few additional sites exhibit shifts that differ from previously reported solution NMR assignments. Nonetheless, these de novo assignments indicate close agreement between the chemical shifts observed in solution and those in microcrystalline or precipitated solids. The methods utilized are likely to be generally applicable for other noncrystalline, nonsoluble proteins.

Carbon Isotopes↗