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Stephen R Martin

Publications and source records attributed to Stephen R Martin.

26 records · Page 2Linked to original sources
PubMed2003

Thermal unfolding simulations of apo-calmodulin using leap-dynamics.

The simulation method leap-dynamics (LD) has been applied to protein thermal unfolding simulations to investigate domain-specific unfolding behavior. Thermal unfolding simulations of the 148-residue protein apo-calmodulin with implicit solvent were performed at temperatures 290 K, 325 K, and 360 K and compared with the corresponding molecular dynamics trajectories in terms of a number of calculated conformational parameters. The main experimental results of unfolding are reproduced in showing the lower stability of the C-domain: at 290 K, both the N- and C-domains are essentially stable; at 325 K, the C-domain unfolds, whereas the N-domain remains folded; and at 360 K, both domains unfold extensively. This behavior could not be reproduced by molecular dynamics simulations alone under the same conditions. These results show an encouraging degree of convergence between experiment and LD simulation. The simulations are able to describe the overall plasticity of the apo-calmodulin structure and to reveal details such as reversible folding/unfolding events within single helices. The results show that by using the combined application of a fast and efficient sampling routine with a detailed molecular dynamics force field, unfolding simulations of proteins at atomic resolution are within the scope of current computational power.

Apoproteins↗
PubMed2002

Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism.

Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.

Adenosine Triphosphate↗
PubMed2002

Temperature jump kinetic study of the stability of apo-calmodulin.

Temperature-jump relaxation spectrometry has been used to study the unfolding properties of Ca(2+)-free Drosophila calmodulin from 278 to 336 K, monitored by absorption of Tyr-138. The T-jump amplitude data are well fitted throughout with a melting temperature T(m) = 315.7 K, deltaH(o)(m) = 140.5 kJ mol(-1) and deltaC(p)(o) = 3.28 kJ K(-1) mol(-1), giving deltaG(o)(293) = 7.36 kJ mol(-1) for the C-domain, in good agreement with other data. The relaxation rate observed (time range 1 micros-1 ms) obeys a simple two-state kinetic mechanism throughout. The activation energy for unfolding is nearly temperature-independent, in contrast to that for refolding, and hence the transition state is relatively compact, resembling the folded state, and the relaxation time, tau, shows complex temperature dependence. The domain unfolding is a two-state process occurring with tau of approximately 100 micros at the T(m). At 296 K, when the C-domain is approximately 6% unfolded, k(unfolding) approximately 305 s(-1), k(refolding) approximately 4660 s(-1) and tau approximately 200 micros. This closely resembles the rate and extent of a reported C-domain exchange process, inferred from NMR line-broadening at 296 K. The inherent instability of the apo-C-domain of calmodulin indicates that the unfolded form significantly contributes to the physical properties of apo-calmodulin at normal temperatures, and this instability is enhanced by low ionic strength conditions.

Animals↗
PubMed2002

Crystal structure and functional analysis of the histone methyltransferase SET7/9.

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.

Amino Acid Sequence↗
PubMed2002

Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins.

Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes.

Animals↗
PubMed2002

Regulatory implications of a novel mode of interaction of calmodulin with a double IQ-motif target sequence from murine dilute myosin V.

Apo-Calmodulin acts as the light chain for unconventional myosin V, and treatment with Ca(2+) can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca(2+) on the stoichiometry and affinity of interactions of calmodulin and its two domains with two myosin-V peptides (IQ3 and IQ4) have therefore been quantified in vitro, using fluorescence and near- and far-UV CD. The results with separate domains show their differential affinity in interactions with the IQ motif, with the apo-N domain interacting surprisingly weakly. Contrary to expectations, the effect of Ca(2+) on the interactions of either peptide with either isolated domain is to increase affinity, reducing the K(d) at physiological ionic strengths by >200-fold to approximately 75 nM for the N domain, and approximately 10-fold to approximately 15 nM for the C domain. Under suitable conditions, intact (holo- or apo-) calmodulin can bind up to two IQ-target sequences. Interactions of apo- and holo-calmodulin with the double-length, concatenated sequence (IQ34) can result in complex stoichiometries. Strikingly, holo-calmodulin forms a high-affinity 1:1 complex with IQ34 in a novel mode of interaction, as a "bridged" structure wherein two calmodulin domains interact with adjacent IQ motifs. This apparently imposes a steric requirement for the alpha-helical target sequence to be discontinuous, possibly in the central region, and a model structure is illustrated. Such a mode of interaction could account for the Ca(2+)-dependent regulation of myosin V in vitro motility, by changing the structure of the regulatory complex, and paradoxically causing calmodulin dissociation through a change in stoichiometry, rather than a Ca(2+)-dependent reduction in affinity.

Amino Acid Motifs↗
PubMedDate not supplied

Fluorescent ligands for the hemagglutinin of influenza A: synthesis and ligand binding assays.

Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a delta-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low microM range. Binding was accompanied by approximately 4.5-fold fluorescence enhancement for the dansyl conjugate 1 and approximately 3-fold fluorescence quenching for the pyrene conjugate 3.

Fluorescent Dyes↗