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Biomedical subjects

A J Wand

Publications and source records attributed to A J Wand.

At least 19 recordsLinked to original sources
PubMed2000

Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex.

The response of the internal dynamics of calcium-saturated calmodulin to the formation of a complex with a peptide model of the calmodulin-binding domain of the smooth muscle myosin light chain kinase has been studied using NMR relaxation methods. The backbone of calmodulin is found to be unaffected by the binding of the domain, whereas the dynamics of side chains are significantly perturbed. The changes in dynamics are interpreted in terms of a heterogeneous partitioning between structure (enthalpy) and dynamics (entropy). These data provide a microscopic view of the residual entropy of a protein in two functional states and suggest extensive enthalpy/entropy exchange during the formation of a protein-protein interface.

Amino Acid Sequence↗
PubMed1999

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Allosteric Site↗
PubMed1999

De novo proteins as models of radical enzymes.

Catalytically essential side-chain radicals have been recognized in a growing number of redox enzymes. Here we present a novel approach to study this class of redox cofactors. Our aim is to construct a de novo protein, a radical maquette, that will provide a protein framework in which to investigate how side-chain radicals are generated, controlled, and directed toward catalysis. A tryptophan and a tyrosine radical maquette, denoted alpha(3)W(1) and alpha(3)Y(1), respectively, have been synthesized. alpha(3)W(1) and alpha(3)Y(1) contain 65 residues each and have molecular masses of 7.4 kDa. The proteins differ only in residue 32, which is the position of their single aromatic side chain. Structural characterization reveals that the proteins fold in water solution into thermodynamically stable, alpha-helical conformations with well-defined tertiary structures. The proteins are resistant to pH changes and remain stable through the physiological pH range. The aromatic residues are shown to be located within the protein interior and shielded from the bulk phase, as designed. Differential pulse voltammetry was used to examine the reduction potentials of the aromatic side chains in alpha(3)W(1) and alpha(3)Y(1) and compare them to the potentials of tryptophan and tyrosine when dissolved in water. The tryptophan and tyrosine potentials were raised considerably when moved from a solution environment to a well-ordered protein milieu. We propose that the increase in reduction potential of the aromatic residues buried within the protein, relative to the solution potentials, is due to a lack of an effective protonic contact between the aromatic residues and the bulk solution.

Amino Acid Sequence↗
PubMed1999

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation.

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau m values for proteins. The near identity of observed T2 and T1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau m values. It is also noted that the reliable determination of tau m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as tau m increases.

Humans↗
PubMed1999

Preparation of encapsulated proteins dissolved in low viscosity fluids.

The majority of proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. One potential approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques is to encapsulate them in a reverse micelle which is dissolved in a low viscosity fluid. Unfortunately, promising low viscosity fluids such as the short chain alkanes, supercritical carbon dioxide, and various halocarbon refrigerants all require the application of significant pressure to be kept liquefied at room temperature. Here we describe the design and use of a simple cost effective NMR tube suitable for the preparation of solutions of proteins encapsulated in reverse micelles dissolved in such fluids.

Capsules↗
PubMed1998

High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids.

The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.

Alkanes↗
PubMed1998

Local stability and dynamics of apocytochrome b562 examined by the dependence of hydrogen exchange on hydrostatic pressure.

Hydrostatic pressure is used to perturb the manifold of states available to apocytochrome b562 and to examine the energetics and dynamics of the protein using hydrogen exchange monitored in real-time by heteronuclear spectroscopy at pressures ranging up to 1. 1 kbar. An analytical framework for interpreting the effects of hydrostatic pressure on the physical events leading to protein hydrogen exchange is presented. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle. The dependence of the global unfolding free energy upon pressure is first order and associated with a negative volume change of -102 mL mol-1. Two additional regions of cooperative structure are identified, and both also have negative volume changes associated with their unfolding at high pressure. Surprisingly, one of the subglobal unfolding units shows a significant positive volume change at low pressures (<200 bar) suggesting the presence of a highly mispacked open state at ambient pressure. The three regions of cooperative stability are the same as identified by perturbation with chemical denaturant. The implications of these results for issues in protein folding and the form of the energy landscape of globular proteins are discussed.

Apoproteins↗
PubMed1998

Local dynamics and stability of apocytochrome b562 examined by hydrogen exchange.

Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this "structured" molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry and hydrogen exchange. These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N- and C-terminal helices fold and unfold independently of the core of the molecule.

Amino Acid Sequence↗
PubMed1998

Determinants of protein hydrogen exchange studied in equine cytochrome c.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.

Animals↗
PubMed1997

Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein.

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-alpha2]2 shows their effective rotational correlation time (tau(c)) is 7.3 +/- 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin-spin interaction between the buried, immobilized spin-labels indicates they are remote (> 15 A) from each other, indicating an antiparallel topology of the monomers in [MAL-6-alpha2]2. The parent [alpha2]2 and the modified [MAL-6-alpha2]2 and [CP-alpha2]2 peptides are highly stable (deltaG(H2O) approximately 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-alpha2]2 to yield two [MAL-6-alpha2] monomers with retention of secondary structure and changed the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-alpha2]2 and [CP-MAL-6-alpha2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-alpha2]2. Consequently, the ESR spectra of [MAL-6-alpha2]2 and [CP-MAL-6-alpha2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-alpha2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha2]2 to be solvent-exposed by addition of [CP-alpha2]2 results in formation of the heterodimeric [CP-alpha2]/[CP-MAL-6-alpha2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

Amino Acid Sequence↗
PubMed1997

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins.

Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters. High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli. This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13C-labeled protein, allowing high-quality measurements on relatively dilute (< 1 mM) protein samples at a relatively low cost.

Bacterial Proteins↗
PubMed1996

Solution structure of horse heart ferricytochrome c and detection of redox-related structural changes by high-resolution 1H NMR.

A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restrains based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY-TOCSY spectra. The all-residue root mean square deviation about the average structure is 0.33 +/- 0.04 A for the backbone N, C alpha, and C' atoms and 0.83 +/- 0.05 A for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes though there are a number of significant differences in detailed aspects of the structure. Two of the three main helices display localized irregularities in helical hydrogen bonding resulting in bifurcation of main chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in reported crystal models. To provide an independent measure of the accuracy of the model for the oxidized protein, the expected pseudocontact shifts induced by the spin 1/2 iron were compared to the observed redox-dependent chemical shift changes. These comparisons confirm the general accuracy of the model for the oxidized protein and its observed differences with the structure of the reduced protein. The structures of the reduced and oxidized states of the protein provide a template to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the cytochrome c molecule. For example, a redox-dependent reorganization of surface residues at the heme edge can be directly related to the redox behavior of the protein and thereby provides a previously undocumented linkage between structural change potentially associated with molecular recognition of redox partners and the fundamental parameters governing electron transfer.

Animals↗
PubMed1996

Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein.

The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism. The generalized order parameters for 56 main chain alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic.

Carbon Isotopes↗
PubMed1996

Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex.

The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than 0.1 M KCl, suggesting that electrostatic interactions contribute strongly to complex formation. Ion pair interactions are frequently sensitive to high hydrostatic pressure due to electrostriction effects in the solvated ion state. Application of high pressure to the apoCaM.Neurop complex causes a red shift of the Neurop tryptophan emission center of mass and a reduced residual anisotropy but with insignificant reduction in quantum yield. The transition is smooth, reversible, and apparently two-state with a midpoint pressure of approximately 0.8 kbar. The residual anisotropy, quantum yield, and center of mass of the emission spectrum are consistent with the movement of the tryptophan side chain to a more solvated, slightly less restricted environment upon the pressure-induced transition. The pressure effect is independent of the concentration of the complex. These and other data are consistent with the pressure-induced reorganization being a unimolecular event not requiring dissociation of the complex. A volume change of approximately 66 mL mol-1 and a free energy change of approximately 1.7 kcal mol-1 are associated with the reorganization. The residual interactions maintaining the complex under high pressure are characterized by low standard molar volume and/or high standard free energy changes upon disruption but are destroyed by 200 mM KCl. It is postulated that the main effect of salt on the complex at high pressure is to drive the collapse of the hydrophobic pocket to which the peptide is binding.

Amino Acid Sequence↗
PubMed1996

Insights into the local residual entropy of proteins provided by NMR relaxation.

A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well-packed extended amino acid side chains are described by employing a one-dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular "model-free" treatment often used in the analysis of NMR relaxation data. Simulations indicate that order parameters observed for the methyl symmetry axes in, for example, human ubiquitin correspond to significant local entropies. These observations have obvious significance for the issue of the physical basis of protein structure, dynamics, and stability.

Entropy↗
PubMed1996

Protein complexes studied by NMR spectroscopy.

Recent advances in NMR methods now allow protein complexes to be studied in great detail in a wide range of solution conditions. Isotope-enrichment strategies, resonance-assignment approaches and structural-determination methods have evolved to the point where almost any type of complex involving proteins of reasonable size may be studied in a straightforward way. A variety of isotope editing and filtering strategies underlie these powerful methodologies. Approaches to the characterization of the dynamics of protein complexes have also matured to the point where detailed studies of the effects of complexation on dynamics can be studied over a wide range of timescales.

Ligands↗
PubMed1995

Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium.

The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.

Amino Acid Sequence↗