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Biomedical subjects

Brian D Sykes

Publications and source records attributed to Brian D Sykes.

At least 19 recordsLinked to original sources
PubMed2003

VADAR: a web server for quantitative evaluation of protein structure quality.

VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.

Algorithms↗
PubMed2003

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C.

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.

Amino Acid Sequence↗
PubMed2003

Structure and dynamics of the C-domain of human cardiac troponin C in complex with the inhibitory region of human cardiac troponin I.

Cardiac troponin C is the Ca2+-dependent switch for heart muscle contraction. Troponin C is associated with various other proteins including troponin I and troponin T. The interaction between the subunits within the troponin complex is of critical importance in understanding contractility. Following a Ca2+ signal to begin contraction, the inhibitory region of troponin I comprising residues Thr128-Arg147 relocates from its binding surface on actin to troponin C, triggering movement of troponin-tropomyosin within the thin filament and thereby freeing actin-binding site(s) for interactions with the myosin ATPase of the thick filament to generate the power stroke. The structure of calcium-saturated cardiac troponin C (C-domain) in complex with the inhibitory region of troponin I was determined using multinuclear and multidimensional nuclear magnetic resonance spectroscopy. The structure of this complex reveals that the inhibitory region adopts a helical conformation spanning residues Leu134-Lys139, with a novel orientation between the E- and H-helices of troponin C, which is largely stabilized by electrostatic interactions. By using isotope labeling, we have studied the dynamics of the protein and peptide in the binary complex. The structure of this inhibited complex provides a framework for understanding into interactions within the troponin complex upon heart contraction.

Amino Acid Sequence↗
PubMed2003

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers.

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289-8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124-31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence of the switch peptide [Gagné et al. (1995) Nat. Struct. Biol. 2, 784-789]. The switch peptide is bound in a shallow and complementary hydrophobic surface cleft largely defined by helices A and B and also has key ionic interactions with sNTnC. These results show that bifunctional rhodamine probes can be attached to surface helices via suitable pairs of solvent-accessible residues that have been mutated to cysteines, without altering the conformation of the labeled domain. A set of such probes can be used to determine the orientation and motion of the target domain in the cellular environment [Corrie et al. (1999) Nature 400, 425-430; Ferguson et al. (2003) Mol. Cell 11(4), in press].

Animals↗
PubMed2003

Lipid-bound structure of an apolipoprotein E-derived peptide.

Apolipoprotein (apo) E regulates plasma lipid homeostasis through its ability to interact with the low density lipoprotein (LDL) receptor family. Whereas apoE is not a ligand for receptor binding in buffer alone, interaction with lipid confers receptor recognition properties. To investigate the nature of proposed lipid binding-induced conformational changes in apoE, we employed multidimensional heteronuclear NMR spectroscopy to determine the structure of an LDL receptor-active, 58-residue peptide comprising residues 126-183 of apoE in association with the micelle-forming lipid dodecylphosphocholine (DPC). In the presence of 34 mm DPC the peptide forms a continuous amphipathic helix from Glu131 to Arg178. NMR relaxation studies of DPC-bound apoE-(126-183), in contrast to apoE-(126-183) in the presence of TFE, are consistent with an isotropically tumbling peptide in solution giving a global correlation time of approximately 12.5 ns. These data indicate that the helical peptide is curved and constrained by a lipid micelle consisting of approximately 48 DPC molecules. Although the peptide behaves as if it were tumbling isotropically, spectral density analysis reveals that residues 150-183 have more motional freedom than residues 134-149. These molecular and dynamic features are discussed further to provide insight into the structural basis for the interaction between apoE and the ligand binding repeats of the LDL receptor.

Amino Acid Sequence↗
PubMed2003

Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature.

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.

Animals↗
PubMed2003

Unmasking ligand binding motifs: identification of a chemokine receptor motif by NMR studies of antagonist peptides.

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.

Amino Acid Motifs↗
PubMed2003

Freezing of a fish antifreeze protein results in amyloid fibril formation.

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.

Amyloid beta-Peptides↗
PubMed2003

PJNMR: a platform-independent graphical simulation tool for NMR spectroscopy.

A new simulation program for multinuclear NMR is introduced. PJNMR (Pure Java NMR) 2.0, written entirely in the Java programming language, simulates pulse sequences on systems of up to three weakly coupled spins-1/2 with a command-driven, spectrometer-like interface. Users may simulate the effects of pulses, precessions, and pulsed field gradients on the spin system, with a graphical display showing the state of the density matrix (in a novel polar-coordinate representation) as well as the magnetization vectors for each nucleus. Relevant computations and optimizations as implemented in the code are detailed, along with the object-oriented structures used. A description of the simulation environment is given, illustrated with a series of example pulse sequences highlighting the insights gained in the graphical presentation.

Algorithms↗
PubMed2003

In situ orientations of protein domains: troponin C in skeletal muscle fibers.

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.

Actins↗
PubMed2003

The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein.

Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the beta-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the (3)J(alphabeta) (1)H-(1)H scalar coupling constants, the chi(1) angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.

Amino Acid Sequence↗
PubMed2003

High-yield expression of isotopically labeled peptides for use in NMR studies.

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.

Amino Acid Sequence↗
PubMed2003

Dynamics of cryoprotectant permeation in porcine heart valve leaflets.

Valve replacement is a common cardiovascular procedure for the treatment of a variety of congenital and acquired defects. Many surgical programs rely on cryopreserved heart valves from regional tissue bank programs to meet clinical demands. Current cryopreservation strategies for heart valves are empirically derived. The aim of this study was to use proton nuclear magnetic resonance spectroscopy (NMR) to monitor changes in cryoprotectant concentration in isolated heart valve leaflets. Porcine aortic valves were locally obtained, freshly isolated, and allowed to equilibrate at various experimental temperatures (22 degrees C, 10 degrees C, 4 degrees C) for 1 h prior to immersion in 1 M Me2SO solution. At defined intervals (0, 0.25, 0.5, 1, 2, 6, and 24 h) the valves were removed from the Me2SO and the leaflets were rapidly dissected and equilibrated in deuterium oxide (D2O). Using previously described techniques the Me2SO concentration in the heart valve leaflets was determined by NMR and the diffusion coefficient was calculated as a function of time and temperature. Heart valve leaflets were fully equilibrated with Me2SO after approximately 2 h of exposure at 22 degrees C while equilibrium was not reached >6 h or more at 10 degrees C and 4 degrees C. These results indicate that that permeation of Me2SO in heart valves is strongly temperature dependent Furthermore, this study provides a quantitative measure of Me2SO permeation and cryoprotectant at equilibration in heart valve leaflets. The clinical applications of these findings may help to optimize the balance between the protective and toxic effects of cryoprotectants and lead to improved methods of preservation of heart valves.

Animals↗
PubMed2002

Identification of the ice-binding face of antifreeze protein from Tenebrio molitor.

The beetle Tenebrio molitor produces several isoforms of a highly disulfide-bonded beta-helical antifreeze protein with one surface comprised of an array of Thr residues that putatively interacts with ice. In order to use mutagenesis to identify the ice-binding face, we have selected an isoform that folds well and is tolerant of amino acid substitution, and have developed a heating test to monitor refolding. Three different types of steric mutations made to the putative ice-binding face reduced thermal hysteresis activity substantially while a steric mutation on an orthogonal surface had little effect. NMR spectra indicated that all mutations affected protein folding to a similar degree and demonstrated that most of the protein folded well. The large reductions in activity associated with steric mutations in the Thr array strongly suggest that this face of the protein is responsible for ice binding.

Amino Acid Sequence↗
PubMed2002

The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions.

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.

Chemokine CXCL10↗
PubMed2002

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C.

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.

Amino Acid Sequence↗
PubMed2002

Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I147-163 and bepridil.

Cardiac troponin C (cTnC) is the Ca(2+)-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Ca(2+) binding to the regulatory domain of cTnC (cNTnC) induces little structural change but sets the stage for cTnI binding. A large "closed" to "open" conformational transition occurs in the regulatory domain upon binding cTnI(147-163) or bepridil. This raises the question of whether cTnI(147-163) and bepridil compete for cNTnC.Ca(2+). In this work, we used two-dimensional (1)H,(15)N-heteronuclear single quantum coherence (HSQC) NMR spectroscopy to examine the binding of bepridil to cNTnC.Ca(2+) in the absence and presence of cTnI(147-163) and of cTnI(147-163) to cNTnC.Ca(2+) in the absence and presence of bepridil. The results show that bepridil and cTnI(147-163) bind cNTnC.Ca(2+) simultaneously but with negative cooperativity. The affinity of cTnI(147-163) for cNTnC.Ca(2+) is reduced approximately 3.5-fold by bepridil and vice versa. Using multinuclear and multidimensional NMR spectroscopy, we have determined the structure of the cNTnC.Ca(2+).cTnI(147-163).bepridil ternary complex. The structure reveals a binding site for cTnI(147-163) primarily located on the A/B interhelical interface and a binding site for bepridil in the hydrophobic pocket of cNTnC.Ca(2+). In the structure, the N terminus of the peptide clashes with part of the bepridil molecule, which explains the negative cooperativity between cTnI(147-163) and bepridil for cNTnC.Ca(2+). This structure provides insights into the features that are important for the design of cTnC-specific cardiotonic drugs, which may be used to modulate the Ca(2+) sensitivity of the myofilaments in heart muscle contraction.

Actin Cytoskeleton↗