Publications by authors named "Piotr G Fajer"

20 Publications

  • Page 1 of 1

The concerted movement of the switch region of Troponin I in cardiac muscle thin filaments as tracked by conventional and pulsed (DEER) EPR.

J Struct Biol 2017 12 31;200(3):376-387. Epub 2017 Aug 31.

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. Electronic address:

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca, the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca, the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2017.08.007DOI Listing
December 2017

Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

PLoS One 2014 13;9(11):e112976. Epub 2014 Nov 13.

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.

The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231091PMC
December 2015

Binding of MgtR, a Salmonella transmembrane regulatory peptide, to MgtC, a Mycobacterium tuberculosis virulence factor: a structural study.

J Mol Biol 2014 Jan 17;426(2):436-46. Epub 2013 Oct 17.

National High Magnetic Field Laboratory, Tallahassee, FL 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA. Electronic address:

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2013.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947350PMC
January 2014

Effects of calcium binding and the hypertrophic cardiomyopathy A8V mutation on the dynamic equilibrium between closed and open conformations of the regulatory N-domain of isolated cardiac troponin C.

Biochemistry 2013 Mar 6;52(11):1950-62. Epub 2013 Mar 6.

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi4000172DOI Listing
March 2013

Interdomain orientation of cardiac troponin C characterized by paramagnetic relaxation enhancement NMR reveals a compact state.

Protein Sci 2012 Sep;21(9):1376-87

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

Cardiac troponin C (cTnC) is the calcium binding subunit of the troponin complex that triggers the thin filament response to calcium influx into the sarcomere. cTnC consists of two globular EF-hand domains (termed the N- and C-domains) connected by a flexible linker. While the conformation of each domain of cTnC has been thoroughly characterized through NMR studies involving either the isolated N-domain (N-cTnC) or C-domain (C-cTnC), little attention has been paid to the range of interdomain orientations possible in full-length cTnC that arises as a consequence of the flexibility of the domain linker. Flexibility in the domain linker of cTnC is essential for effective regulatory function of troponin. We have therefore utilized paramagnetic relaxation enhancement (PRE) NMR to assess the interdomain orientation of cTnC. Ensemble fitting of our interdomain PRE measurements reveals that isolated cTnC has considerable interdomain flexibility and preferentially adopts a bent conformation in solution, with a defined range of relative domain orientations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.2124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631366PMC
September 2012

Improved sequence resolution by global analysis of overlapped peptides in hydrogen/deuterium exchange mass spectrometry.

J Am Soc Mass Spectrom 2012 Jul 24;23(7):1202-8. Epub 2012 Apr 24.

Institute of Molecular Biophysics, Biological Sciences Department, Florida State University, Tallahassee, FL 32306, USA.

Management of the enormous amount of data produced during solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has stimulated software analysis development. The proteolysis step of the experiment generates multiple peptide fragments, most of which overlap. Prior automated data reduction algorithms extract the deuteration level for individual peptides, but do not exploit the additional information arising from fragment overlap. Here, we describe an algorithm that determines discrete rate constant values to each of the amide hydrogens in overlapped fragments. By considering all of the overlapped peptide segments simultaneously, sequence resolution can be improved significantly, sometimes to the individual amino acid level. We have validated the method with simulated deuterium uptake data for seven overlapped fragments of a poly-Ala nonapeptide, and then applied it to extract rate constant values for the first 29 N-terminal amino acids of C22A FK506-binding protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13361-012-0373-3DOI Listing
July 2012

Complexation and Calcium-Induced Conformational Changes in the Cardiac Troponin Complex Monitored by Hydrogen/Deuterium Exchange and FT-ICR Mass Spectrometry.

Int J Mass Spectrom 2011 Apr;302(1-3):116-124

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306.

Cardiac muscle contraction is regulated by the heterotrimeric complex: troponin. We apply solution-phase hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry to study the structural dynamics and the Ca-induced conformational changes of the cardiac isoform of troponin, by comparing H/D exchange rate constants for TnC alone, the binary TnC:TnI complex, and the ternary TnC:TnI:TnT complex for Ca-free and Ca-saturated states. The wide range of exchange rate constants indicates that the complexes possess both highly flexible and very rigid domains. Fast exchange rates were observed for the N-terminal extension of TnI (specific to the cardiac isoform), the DE linker in TnC alone, and the mobile domain of TnI. The slowest rates were for the IT coiled-coil that grants stability and stiffness to the complex. Ca(2+) binding to site II of the N-lobe of TnC induces short-range allosteric effects, mainly protection for the C-lobe of TnC that transmits long-range conformational changes that reach the IT coiled-coil and even TnT1. The present results corroborate prior X-ray crystallography and NMR interpretations and also illuminate domains that were not resolved or truncated in those experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijms.2010.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3134279PMC
April 2011

Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe.

PLoS One 2011 20;6(6):e21277. Epub 2011 Jun 20.

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118794PMC
October 2011

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.

Proc Natl Acad Sci U S A 2011 May 2;108(20):8218-23. Epub 2011 May 2.

National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, USA.

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1014137108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100986PMC
May 2011

Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry.

Anal Chem 2010 Apr;82(8):3293-9

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of (13)C and/or (15)N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ((12)C(c)(1)H(h)(14)N(n)(16)O(o)(32)S(s)), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac100079zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878611PMC
April 2010

Distance and dynamics determination by W-band DEER and W-band ST-EPR.

Eur Biophys J 2010 Mar 9;39(4):711-9. Epub 2009 Dec 9.

National High Magnetic Field Laboratory, Institute of Molecular Biophysics, Department of Biological Science, Department of Biochemistry and Chemistry, Florida State University, Tallahassee, FL 32310, USA.

To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-009-0565-3DOI Listing
March 2010

Nucleotide-induced flexibility change in neck linkers of dimeric kinesin as detected by distance Measurements using spin-labeling EPR.

J Mol Biol 2009 Feb 6;386(3):626-36. Epub 2009 Jan 6.

Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, approximately 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5'-(beta,gamma-imido)triphosphate and adenosine 5'-(gamma-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered. We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2008.12.079DOI Listing
February 2009

Protein comparative sequence analysis and computer modeling.

Methods Mol Med 2008 ;141:245-56

Pathology Discipline, Bosch Institute, School of Medical Science, University of Sydney, New South Wales, Australia.

A problem frequently encountered by the biological scientist is the identification of a previously unknown gene or protein sequence, where there are few or no clues as to the biochemical function, ligand specificity, gene regulation, protein-protein interactions, tissue specificity, cellular localization, developmental phase of activity, or biological role. Through the process of bioinformatics there are now many approaches for predicting answers to at least some of these questions, often then allowing the design of more insightful experiments to characterize more definitively the new protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60327-148-6_13DOI Listing
June 2008

Mapping electron paramagnetic resonance spin label conformations by the simulated scaling method.

J Am Chem Soc 2007 Nov 19;129(45):13840-6. Epub 2007 Oct 19.

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA.

In order to efficiently simulate spin label behavior when attached to the protein backbone we developed a novel approach that enhances local conformational sampling. The simulated scaling (SS) approach (Li, H., et al. J. Chem. Phys. 2007, 126, 24106) couples the random walk of a potential scaling parameter and molecular dynamics in the framework of hybrid Monte Carlo. This approach allows efficient barrier crossings between conformations. The method retains the thermodynamic detailed balance allowing for determination of relative free energies between various conformations. The accuracy of our method was validated by comparison with the recently resolved X-ray crystal structure of a spin labeled T4 lysozyme in which the spin label was in the interior of the protein. Consistent potentials of mean force (PMF) are obtained for the spin label torsion angles to illustrate their behavior in various protein environments: surface, semiburied, and buried. These PMFs reflect the experimentally observed trends and provide the rationale for the spin label dynamics. We have used this method to compare an implicit and explicit solvent model in spin label modeling. The implicit model, which is computationally faster, was found to be in excellent agreement with the explicit solvent treatment. Based on this collection of results, we believe that the presented approach has great potential in the general strategy of describing the behavior of the spin label using molecular modeling and using this information in the interpretation of EPR measurements in terms of protein conformation and dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja071404vDOI Listing
November 2007

Protein dynamics and monomer-monomer interactions in AntR activation by electron paramagnetic resonance and double electron-electron resonance.

Biochemistry 2007 Oct 19;46(41):11639-49. Epub 2007 Sep 19.

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA.

The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi700859pDOI Listing
October 2007

Regulatory and catalytic domain dynamics of smooth muscle myosin filaments.

Biochemistry 2006 May;45(19):6212-21

Biology Department, Institute of Molecular Biophysics and the National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA.

Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi060037hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090715PMC
May 2006

Mn(II) binding by the anthracis repressor from Bacillus anthracis.

Biochemistry 2006 Apr;45(13):4295-303

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA.

The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 microM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s(-1). Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi052288gDOI Listing
April 2006

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods.

Biophys J 2004 May;86(5):3030-41

Department of Biological Sciences, Florida State University, Tallahassee, Florida, USA.

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304169PMC
http://dx.doi.org/10.1016/S0006-3495(04)74352-0DOI Listing
May 2004

Myosin cleft movement and its coupling to actomyosin dissociation.

Nat Struct Biol 2003 Oct 21;10(10):831-5. Epub 2003 Sep 21.

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsb986DOI Listing
October 2003

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance.

Proc Natl Acad Sci U S A 2002 Oct 18;99(20):12765-70. Epub 2002 Sep 18.

National High Magnetic Field Laboratory, Institute of Molecular Biophysics, and Department of Biological Science, Florida State University, Tallahassee, FL 32310, USA.

Site-directed spin labeling EPR (SDSL-EPR) was used to determine the structure of the inhibitory region of TnI in the intact cardiac troponin ternary complex. Maeda and collaborators have modeled the inhibitory region of TnI (skeletal 96-112: the structural motif that communicates the Ca(2+) signal to actin) as a kinked alpha-helix [Vassylyev, D., Takeda, S., Wakatsuki, S., Maeda, K. & Maeda, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 4847-4852), whereas Trewhella and collaborators have proposed the same region to be a flexible beta-hairpin [Tung, C. S., Wall, M. E., Gallagher, S. C. & Trewhella, J. (2000) Protein Sci. 9, 1312-1326]. To distinguish between the two models, residues 129-145 of cardiac TnI were mutated sequentially to cysteines and labeled with the extrinsic spin probe, MTSSL. Sequence-dependent solvent accessibility was measured as a change in power saturation of the spin probe in the presence of the relaxation agent. In the ternary complex, the 129-137 region followed a pattern characteristic of a regular 3.6 residues/turn alpha-helix. The following region, residues 138-145, showed no regular pattern in solvent accessibility. Measurements of 4 intradomain distances within the inhibitory sequence, using dipolar EPR, were consistent with an alpha-helical structure. The difference in side-chain mobility between the ternary (C.I.T) and binary (C.I) complexes revealed a region of interaction of TnT located at the N-terminal end of the inhibitory sequence, residues 130-135. The above findings for the troponin complex in solution do not support either of the computational models of the binary complex; however, they are in very good agreement with a preliminary report of the x-ray structure of the cardiac ternary complex [Takeda, S. Yamashita, A., Maeda, K. & Maeda, Y. (2002) Biophys. J. 82, 832].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.202477399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130534PMC
October 2002