ANSTO :: Browsing by Author "Trewhella, J"

Browsing by Author "Trewhella, J"

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The ANSTO contribution to a project to provide experimental standards for SAS profile prediction
(Australian Institure of Nuclear Sicence and Engineering (AINSE), 2020-11-11) Duff, AP; Kirby, N; Ryan, T; Trewhella, J; Whitten, AE; Wood, K
The program CRYSOL1, was the first method developed to rapidly calculate small-angle scattering (SAS) profiles from atomic coordinates of biomolecules. It was a major breakthrough, providing the missing link between high-resolution structures and solution SAS data. The importance of this breakthrough is evident in the fact that decades later, alternate methods continue to be published with various claims of improvement. To date, each of the alternate methods published have been validated using different data sets and models. A consensus set of high quality data would be of considerable value in benchmarking the different approaches. In order to evaluate different approaches to including the hydration layer contribution to the SAS profile, it is desirable to have data obtained using X-rays (SAXS) and neutrons (SANS), the latter in H2O and D2O, as the hydration layer contribution differs significantly for each of these measurements. To develop a consensus set of high quality data, an international project involving the efforts of 37 researchers from 11 different X-ray and 3 different neutron scattering facilities across Asia, Europe and North Ⓒ The Authors.
Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release
(Cell Press, 2012-08-08) Taylor, JEN; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, J
Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ∼70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca2+ or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca2+-signaling during viral processing. © 2012 Biophysical Society.
Calmodulin disrupts the structure of the HIV-1 MA protein
(Elsevier, 2010-07-23) Chow, JYH; Jeffries, CM; Kwan, AH; Guss, JM; Trewhella, J
The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks, MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium-sensing calmodulin (CaM), which is up-regulated upon HIV-1 infection. The nature of the CaM–MA interaction has been the subject of structural studies, using peptides based on the MA sequence, that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca2+-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two buried tryptophans (W16 and W36) located in the first two alpha-helices of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, whereas chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the normal target protein-binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N- and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA loses ~ 20% of its α-helical content upon CaM binding. Thus, CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions. © 2010, Elsevier Ltd.
Cardiac myosin-binding protein C decorates F-actin: implications for cardiac function
(National Academy of Sciences, 2008-11-25) Whitten, AE; Jeffries, CM; Harris, SP; Trewhella, J
Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. © 2008, National Academy of Sciences
Changes in small-angle x-ray scattering parameters observed upon binding of ligand to rabbit muscle pyruvate kinase are not correlated with allosteric transitions
(American Chemical Society, 2010-08-24) Fenton, AW; Williams, R; Trewhella, J
Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M-1-PYK). In the absence of substrate [phosphoenolpyruvate (PEP)], SAXS-monitored conformational changes in M-1-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M-1-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under our assay conditions, these small amino acids bind to the protein but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, we can conclude that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M-1-PYK small amino acid complexes (i.e., the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme versus the M-1-PYK small amino acid complexes. © 2010, American Chemical Society
A chemically modified alpha-amylase with a molten-globule state has entropically driven enhanced thermal stability
(Oxford University Press, 2010-10-01) Siddiqui, KS; Poljak, A; Francisci, D; Guerriero, G; Pilak, O; Burg, D; Raftery, MJ; Parkin, DM; Trewhella, J; Cavicchioli, R
The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA(MOD) exhibited a 200% improvement in starch-hydrolyzing productivity at 60 degrees C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAAMOD had formed a thermostable, MG state, in which the unfolding of the tertiary structure preceded that of the secondary structure by at least 20 degrees C. The X-ray crystal structure of TAAMOD revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAAMOD, we rationalised that thermostabilisation have been caused by a decrease in the entropy of the transition state, rather than being enthalpically driven. Far-UV CD shows that the origin of decreased entropy may have arisen from a higher helical content of TAAMOD. This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.© 2010, Oxford University Press (OUP)
Different views from small angles.
(National Academy of Sciences, 2008-04-01) Trewhella, J
The small-angle scattering of x-rays or neutrons from proteins in solution can provide important information about the structure of the protein and the nature of interactions or distance correlations among the protein molecules (1, 2). The former is encoded in the form factor [P(q)], and the latter in the structure factor [S(q)]. These functions are of great interest to the structural biology community; the form factor can be used to develop and test three-dimensional structural models of proteins (3, 4), whereas the structure factor can inform efforts to crystallize proteins for high-resolution structural analysis by providing insights into their organization in solution (4, 5). Because the measured small-angle scattering profile [I(q)] from a solution of particles is proportional to the product of the ensemble and rotationally averaged form factor and the structure factor [P(q)S(q)], accurate extraction of the two contributing functions, and their subsequent interpretation, is complex. The work of Shukla et al. reported in this issue of PNAS (6) is aimed at resolving controversy with regard to the interpretation of small-angle scattering data from solutions of the well studied protein lysozyme. The specific issue examined concerns the interpretation of the extracted S(q) in terms of intermolecular interactions among lysozyme molecules. The conclusion drawn by Shukla et al. contradicts previously published interpretations of similar data and is boldly presented as their title: "Absence of equilibrium cluster phase in concentrated lysozyme solutions." © 2008, National Academy of Sciences
Domain organization of the monomeric form of the Tom70 mitochondrial import receptor
(Elsevier, 2009-05-22) Mills, RD; Trewhella, J; Qiu, TW; Welte, T; Ryan, TM; Hanley, TL; Knott, RB; Lithgow, T; Mulhern, TD
Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical Ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this "open" model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side oil one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS Was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition. © 2009, Elsevier Ltd.
Effect of temperature during assembly on the structure and mechanical properties of peptide-based materials.
(American Chemical Society, 2010-06-01) Ramachandran, S; Taraban, MB; Trewhella, J; Gryczynski, I; Gryczynski, Z; Yu, YB
Mutually complementary, self-repulsive oligopeptide pairs were designed to coassemble into viscoelastic hydrogels. Peptide engineering was combined with biophysical techniques to investigate the effects of temperature on the structural and mechanical properties of the resulting hydrogels. Biophysical characterizations, including dynamic rheometry, small-angle X-ray scattering (SAXS), and fluorescence spectroscopy, were used to investigate hydrogelation at the bulk, fiber, and molecular levels, respectively. It has been found that temperature has a significant effect on the structure and mechanical properties of peptide-based biomaterials. Oligopeptide fibers assembled at 25°C are formed faster and are two times thicker, and the resulting material is mechanically seven times stronger than that assembled at 5°C. © 2010, American Chemical Society
Effects of chain length on oligopeptide hydrogelation
(Royal Society of Chemistry, 2011-03-21) Taraban, MB; Ramachandran, S; Gryczynski, I; Gryczynski, Z; Trewhella, J; Yu, YB
The co-assembly of mutually complementary, but self-repulsive oligopeptide pairs into viscoelastic hydrogels has been studied. Oligopeptides of 6, 10, and 14 amino acid residues were used to investigate the effects of peptide chain length on the structural and mechanical properties of the resulting hydrogels. Biophysical characterizations, including dynamic rheometry, small-angle X-ray scattering (SAXS) and fluorescence spectroscopy, were used to investigate hydrogelation at the bulk, fiber, and molecular levels, respectively. Upon mixing, the 10-mer peptides and the 14-mer peptides both form hydrogels while the 6-mer peptides do not. SAXS studies point to morphological similarity of the cross-sections of fibers underlying the 10 : 10 and 14 : 14 gels. However, fluorescence spectroscopy data suggest tighter packing of the amino acid side chains in the 10 : 10 fibers. Consistent with this tighter packing, dynamic rheometry data show that the 10 : 10 gel has much higher elastic modulus than the 14 : 14-mer (18 kPa vs. 0.1 kPa). Therefore, from the standpoint of mechanical strength, the optimum peptide chain length for this class of oligopeptide-based hydrogels is around 10 amino acid residues. © 2010, Royal Society of Chemistry.
Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching
(Elsevier (Cell Press), 2011-02-16) Johansen, D; Jeffries, CM; Hammouda, B; Trewhella, J; Goldenberg, DP
Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage lambda, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D2O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions. © 2011, Cell Press
Evolution of quaternary structure in a homotetrameric enzyme
(Elsevier, 2008-07-18) Griffin, MDW; Dobson, RCJ; Pearce, FG; Antonio, L; Whitten, AE; Liew, K; Mackay, JP; Trewhella, J; Jameson, GB; Perugini, MA; Gerrard, JA
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits. © 2008, Elsevier Ltd.
Invited review: probing the structures of muscle regulatory proteins using small-angle solution scattering
(Wiley-Blackwell, 2011-08-01) Lu, YL; Jeffries, CM; Trewhella, J
Small-angle X-ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small-angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 505-516, 2011.
K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity
(Oxford University Press, 2012-11-15) Mokbel, N; Ilkovski, B; Kreissl, M; Memo, M; Jeffries, CM; Marttila, M; Lehtokari, VL; Lemola, E; Grönholm, M; Yang, N; Menard, D; Marcorelles, P; Echaniz-Laguna, A; Reimann, J; Vainzof, M; Monnier, N; Ravenscroft, G; McNamara, E; Nowak, KJ; Laing, NG; Wallgren-Pettersson, C; Trewhella, J; Marston, S; Ottenheijm, C; North, KN; Clarke, NF
Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity. © 2012, Oxford University Press
Ligand-induced conformational changes and conformational dynamics in the solution structure of the lactose repressor protein
(Elsevier, 2008-02-15) Taraban, M; Zhan, HL; Whitten, AE; Langley, DB; Matthews, KS; Swint-Kruse, L; Trewhella, J
We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer-homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer-oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or subdomains within the structure. The binding of IPTG to native (tetrameric) LacI-oDNA complexes also appears to facilitate a modest change in the average homodimer-homodimer disposition. Notably, the crystal structure of the native LacI-oDNA complex differs significantly from the average solution conformation. The solution scattering data are best fit by an ensemble of structures that includes (1) similar to 60% of the V-shaped dimer of homodimers observed in the crystal structure and (2) similar to 40% of molecules with more "open" forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain: In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA-binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression. © 2007, Elsevier Ltd.
Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor.
(Elsevier, 2007-11-09) Chan, J; Whitten, AE; Jeffries, CM; Bosanac, I; Mal, TK; Ito, J; Porumb, H; Michikawa, T; Mikoshiba, K; Trewhella, J; Ikura, M
Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP3) receptor (IP3), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP3R have been reported; those of the IP3-binding core (IP3RCORE) with bound IP3, and the suppressor domain. This study examines the structural effects of ligand binding on an IP3R construct, designated IP3RN, that contains both the IP3-binding core and the suppressor domain. Our circular dichroism results reveal that the IP3- bound and IP3-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP3 has a large effect on the stability of IP3RCORE, it has little effect on IP3RN, indicating that the suppressor domain is critical to the stability of IP3RN. The NMR data for IP3RN provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP3-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP3RN undergoes a change m average conformation in response to IP3-binding and the presence of Ca2+, in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of lP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP3 binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set. © 2007, Elsevier Ltd.
LIM domain binding proteins 1 and 2 have different oligomeric states
(Elsevier, 2010-05-28) Cross, AJ; Jeffries, CM; Trewhella, J; Matthews, JM
LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14–200 and 21–197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb114–200 forms a trimer and Ldb221–197 undergoes a monomer–tetramer–octamer equilibrium, where the association in each case is of moderate affinity (105 M− 1). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes. © 2010, Elsevier Ltd.
Macromolecular architecture of extracellular domain of αNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition.
(Elsevier (Cell Press), 2010-08-11) Comoletti, D; Miller, MT; Jeffries, CM; Wilson, J; Demeler, B; Taylor, P; Trewhella, J; Nakagawa, T
Neurexins are multidomain synaptic cell-adhesion proteins that associate with multiple partnering proteins. Genetic evidence indicates that neurexins may contribute to autism, schizophrenia, and nicotine dependence. Using analytical ultracentrifugation, single-particle electron microscopy, and solution X-ray scattering, we obtained a three-dimensional structural model of the entire extracellular domain of neurexin-1α. This protein adopts a dimensionally asymmetric conformation that is monomeric in solution, with a maximum dimension of ~ 170 Å. The extracellular domain of α-neurexin maintains a characteristic “Y” shape, whereby LNS domains 1–4 form an extended base of the “Y” and LNS5-6 the shorter arms. Moreover, two major regions of flexibility are present: one between EGF1 and LNS2, corresponding to splice site 1, another between LNS5 and 6. We thus provide the first structural insights into the architecture of the extracellular region of neurexin-1α, show how the protein may fit in the synaptic cleft, and how partnering proteins could bind simultaneously. © 2010, Cell Press
The motif of human cardiac myosin-binding protein C is required for its Ca2+-dependent Interaction with calmoduli
(American Society for Biochemistry and Molecular Biology, 2012-09-07) Lu, YL; Kwan, AH; Jeffries, CM; Guss, JM; Trewhella, J
The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin Delta S2. Here we report a novel Ca2+-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca2+/CaM with a moderate affinity (K-D similar to 10 mu M), which is similar to the affinity previously determined for myosin Delta S2. However, unlike the interaction with myosin Delta S2, the Ca2+/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca2+/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca2+ dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca2+ binding. Overall, Ca2+/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca2+ signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction. © 2012, American Society for Biochemistry and Molecular Biology.
MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies.
(Wiley-Blackwell, 2008-02) Whitten, AE; Ca, SZ; Trewhella, J
Small-angle neutron scattering with contrast variation can fill important gaps in our understanding of biomolecular assemblies, providing constraints that can aid in the construction of molecular models and in subsequent model refinements. This paper describes the implementation of simple tools for analysing neutron contrast variation data, accessible via a user-friendly web-based interface (http://www.mmb.usyd.edu.au/NCVWeb/). There are three modules accessible from the website to analyse neutron contrast variation data from bimolecular complexes. The first module, Contrast, computes neutron contrasts of each component of the complex required by the other two modules; the second module, R, analyses the contrast dependence of the radii of gyration to yield information relating to the size and disposition of each component in the complex; and the third, Compost, decomposes the contrast variation series into composite scattering functions, which contain information regarding the shape of each component of the complex, and their orientation with respect to each other. © 2008, Wiley-Blackwell.

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