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Molecular Insights into Biomolecular Structure and Dynamics by 14N NMR
Tuesday, February 14, 2017 conformation, while Ca induces a shift in equilibrium toward two additional conformations, one more open and another more closed (‘‘compact’’). Addition of RyRp caused the two lobes of CaM to collapse toward each other (compact conformation), while exhibiting a broad distribution, indicating high flexibility. Removal of Ca from the complex caused increased distance between the spin labels, indicating greater lobe separation. Distances measured between BSL (on CaM) and TOAC (on RyRp) confirmed the compact but flexible conformation of the complex. These results provide insight into the structural dynamics of the Ca-dependent regulation of RyR by CaM. 2196-Pos Board B516 High-Resolution Structural Dynamics of Bifunctionally Spin-Labeled Myosin by EPR of Oriented Muscle Fibers Yahor Savich, Benjamin P. Binder, Peter D. Martin, Andrew R. Thompson, David D. Thomas. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. We have performed electron paramagnetic resonance (EPR) on actin-bound myosin labeled with a bifunctional spin label (BSL), in oriented muscle fibers. Our goal was to determine the effect of known small-molecule effectors on the structure of the complex of the myosin II catalytic domain (CD) bound to actin. The use of BSL in our EPR experiments greatly enhances the resolution of orientation and interspin distance measurements, due to the stereospecific bifunctional attachment to the protein backbone at two engineered Cys residues. We used Dictyostelium myosin II with Cys labeling sites as a model. Three pairs of Cys residues were engineered on our construct, which were located on relay helix, K-helix (upper 50 kDa domain), and W-helix (lower 50 kDa domain). Skinned muscle fiber bundles (actin filaments), decorated with spin-labeled myosin in the absence of nucleotide (‘‘rigor’’), were oriented parallel to the spectrometer’s applied magnetic field. This procedure enables measurement of the orientation of BSL with respect to actin, which in turn gives us the orientation of individual myosin II structural elements. We tested several known myosin effectors, including arachidonic acid (AA). We observed a significant change in the structure of actin-bound myosin induced by AA, consistent with a change in the actin-activated ATPase activity of the spin-labeled construct. Our goal is to understand the structural changes in the myosin II CD in the presence of activators and inhibitors of actomyosin. This work was supported by NIH grant AR32961 to DDT. 2197-Pos Board B517 The Alpha-Synuclein Fibril Fold - Comparing Models from Electron Paramagnetic Resonance and NMR Pravin Kumar1, Maryam Hashemi Shabestari1, Nathalie Schilderink2, Ine M.J. Segers-Nolten2, Mireille M.A.E. Claessens2, Vinod Subramaniam3, Martina Huber1. 1 Leiden University, Department of Physics, Huygens-Kamerlingh Onnes Lab, Leiden, Netherlands, 2University of Twente, Nanobiophysics, MESAþ Institute for Nanotechnology & MIRA Institute for Biomedical Technology and Technical Medicine, Enschede, Netherlands, 3FOM Institute AMOLF & Vrije Universiteit Amsterdam, Nanobiophysics, MESAþ Institute for Nanotechnology, University of Twente & FOM Institute AMOLF, Amsterdam, & Vrije Universiteit of Amsterdam, Amsterdam, Netherlands. Amyloid fibrils and plaques are hallmarks of neurodegenerative diseases. In Parkinson’s disease, plaques (Lewy bodies) consist predominantly of the a-synuclein (aS) protein. To understand aggregation and elucidate the role of mutants in the disease, the molecular architecture of aS fibrils needs to be known. By double electron-electron paramagnetic resonance (DEER), nmdistance constraints can be determined. This is done by DEER on fibrils of doubly spin-labeled aS-variants, diamagnetically diluted with wild-type aS to suppress intermolecular interactions. Intramolecular distances in three pairs (56/69, 56/90 and 69/90) are reported. An approach to derive a model for the fibril-fold from sparse distance data assuming parallel b-sheets is described. Using the DEER distances as input, a model was derived with three strands, comprising residues 56 to 90, in which the strands consist of eight to twelve residues each. Details are described in[1], where also the viability of such a simple model is discussed. Comparison to structural models in the literature given in[1] is augmented in the present contribution by the discussion of the recently published NMR structure of aS fibrils.[2] This structure enables us to compare our constraints directly with the NMR-derived structure, using the MMM model for spin-label attachment.[3] [1] Hashemi Shabestari et al. Appl Magn Reson 46, 369 (2015) DOI 10.1007/ s00723-014-0622-7 [2] Tuttle et al. Nat Struct Mol Biol. 23, 409 (2016) [3] Polyhach et al. Physical Chemistry Chemical Physics 13 2356 (2011)
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2198-Pos Board B518 Structural Dimerization Analysis of the G44V CrgA Mutant from the M. Tuberculosis Divisome Joshua A. Taylor1, Haujun Qin2, Yisuel Shin1, Krishna Sarva3, Malini Rajagopalan3, Timothy Cross1,2. 1 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA, 2NHMFL FSU, Tallahassee, FL, USA, 3Health Science Center, University of Texas, Tyler, TX, USA. CrgA is a 93 amino acid residue protein with two transmembrane helices. It is thought that CrgA plays an important role in the formation of the divisome complex for Mycobacterium tuberculosis by recruiting other proteins to the divisome. Previous research has suggested that CrgA strongly interacts with FtsZ, FtsQ, CwsA, and Penicillin-binding proteins, PBPA and FtsI. In particular, CrgA is likely to play a key role in the activation and maintenance of FtsI in the divisome. While the functional role of CrgA with these other proteins is still being investigated, efforts to structurally characterize this protein has led to the discovery that CrgA appears to be functioning as a dimer based on 2-hybrid assays. Mutagenesis was conducted on the CrgA sequence, leading to the development of several new constructs. Among these, two mutants are of special interest with the mutation of Glycine-44 to Valine and Alanine-78 to Valine resulting in increased stability of the dimer. Based on SDS-PAGE gels, the G44V mutant was seen as the most dramatically stabilized dimer. Usually, glycine residues, when exposed to the fatty acyl environment of a lipid bilayer, facilitates binding. Consequently, this mutant is of particular interest, not only for its role in Mtb but also pertaining to membrane protein biophysics. This structure is now being characterized through solid state nuclear magnetic resonance (ssNMR) spectroscopy. In particular, Magic Angle Spinning (MAS) spectroscopy is being employed to measure inter-helical and inter-monomer distance restraints with the protein in liquid crystalline liposomes of POPC and POPG with differentially-13C labeled monomers. The results of these experiments will be described. 2199-Pos Board B519 Molecular Insights into Biomolecular Structure and Dynamics by 14N NMR Maria Concistre1, James A. Jarvis2, Ibraheem M. Haies1, Ilya Kuprov1, Marina Carravetta1, Philip T.F. Williamson2. 1 Chemistry Department, University of Southampton, Southampton, United Kingdom, 2Biological Sciences, University of Southampton, Southampton, United Kingdom. The structural and dynamic analysis of proteins by NMR in the solid-state has typically required extensive isotope labelling. Here we report our progress using the naturally occurring isotopes 1H and 14N to probe the structure and dynamics of biomolecules. Employing indirect-detection methods, we have previously demonstrated the feasibility of characterising the quadrupolar interaction present at the naturally occurring 14N sites within proteins using 13C as a ‘spy’ nucleus (Jarvis, 2013). These methods demonstrated that in well folded proteins, limited dynamic averaging of the quadrupolar interaction occurred and the magnitude of the quadrupolar interaction reflected the nature of the hydrogen bonding experienced by the nitrogen, with differences of up to 200 kHz measured between amides in alpha helices and beta sheets - allowing a detailed analysis of the electronic environment along the protein backbone. We have extended this methodology to exploit protons as a spy nucleus, where significant enhancements in sensitivity are realised. These advances have opened up the possibility of conducting a molecular analysis of unlabelled biomolecules such as ex-vivo or environmental samples. Currently, we are applying these methods to amyloid fibres, to provide insights into their backbone conformation and polymorphism. Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisation of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. 15(20): p. 7613-20. 2200-Pos Board B520 Structural Determination of a Peptide Residue of a Protein of Immunological Interest NMR Diego M. Lo´pez1, Adriana J. Bermudez2, Yuly E. Sa´nchez1. 1 Physics, National University of Colombia, Bogota, Colombia, 2 Fundacio´n Instituto Inmunologico DE Colombia, Bogota, Colombia. The X-ray difraction and the nuclear magnetic recsonance (NMR) are the most comun methos for the determination of the tridimentional structure of peptides and proteins. The nmr allows to know the structure of the molecule simulating their natural environment, which does not allow X-ray difraction, it gives us a more accurate idea of the molecular estructure, because these structures show a dinamic due to the flexibility of the lateral chains. This paper aims show the process of determining the structure of a peptide residue of the Myelin basic protein (MBP) simulating their natural environment, through the use of NMR.
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2198-Pos Board B518 Structural Dimerization Analysis of the G44V CrgA Mutant from the M. Tuberculosis Divisome Joshua A. Taylor1, Haujun Qin2, Yisuel Shin1, Krishna Sarva3, Malini Rajagopalan3, Timothy Cross1,2. 1 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA, 2NHMFL FSU, Tallahassee, FL, USA, 3Health Science Center, University of Texas, Tyler, TX, USA. CrgA is a 93 amino acid residue protein with two transmembrane helices. It is thought that CrgA plays an important role in the formation of the divisome complex for Mycobacterium tuberculosis by recruiting other proteins to the divisome. Previous research has suggested that CrgA strongly interacts with FtsZ, FtsQ, CwsA, and Penicillin-binding proteins, PBPA and FtsI. In particular, CrgA is likely to play a key role in the activation and maintenance of FtsI in the divisome. While the functional role of CrgA with these other proteins is still being investigated, efforts to structurally characterize this protein has led to the discovery that CrgA appears to be functioning as a dimer based on 2-hybrid assays. Mutagenesis was conducted on the CrgA sequence, leading to the development of several new constructs. Among these, two mutants are of special interest with the mutation of Glycine-44 to Valine and Alanine-78 to Valine resulting in increased stability of the dimer. Based on SDS-PAGE gels, the G44V mutant was seen as the most dramatically stabilized dimer. Usually, glycine residues, when exposed to the fatty acyl environment of a lipid bilayer, facilitates binding. Consequently, this mutant is of particular interest, not only for its role in Mtb but also pertaining to membrane protein biophysics. This structure is now being characterized through solid state nuclear magnetic resonance (ssNMR) spectroscopy. In particular, Magic Angle Spinning (MAS) spectroscopy is being employed to measure inter-helical and inter-monomer distance restraints with the protein in liquid crystalline liposomes of POPC and POPG with differentially-13C labeled monomers. The results of these experiments will be described. 2199-Pos Board B519 Molecular Insights into Biomolecular Structure and Dynamics by 14N NMR Maria Concistre1, James A. Jarvis2, Ibraheem M. Haies1, Ilya Kuprov1, Marina Carravetta1, Philip T.F. Williamson2. 1 Chemistry Department, University of Southampton, Southampton, United Kingdom, 2Biological Sciences, University of Southampton, Southampton, United Kingdom. The structural and dynamic analysis of proteins by NMR in the solid-state has typically required extensive isotope labelling. Here we report our progress using the naturally occurring isotopes 1H and 14N to probe the structure and dynamics of biomolecules. Employing indirect-detection methods, we have previously demonstrated the feasibility of characterising the quadrupolar interaction present at the naturally occurring 14N sites within proteins using 13C as a ‘spy’ nucleus (Jarvis, 2013). These methods demonstrated that in well folded proteins, limited dynamic averaging of the quadrupolar interaction occurred and the magnitude of the quadrupolar interaction reflected the nature of the hydrogen bonding experienced by the nitrogen, with differences of up to 200 kHz measured between amides in alpha helices and beta sheets - allowing a detailed analysis of the electronic environment along the protein backbone. We have extended this methodology to exploit protons as a spy nucleus, where significant enhancements in sensitivity are realised. These advances have opened up the possibility of conducting a molecular analysis of unlabelled biomolecules such as ex-vivo or environmental samples. Currently, we are applying these methods to amyloid fibres, to provide insights into their backbone conformation and polymorphism. Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisation of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. 15(20): p. 7613-20. 2200-Pos Board B520 Structural Determination of a Peptide Residue of a Protein of Immunological Interest NMR Diego M. Lo´pez1, Adriana J. Bermudez2, Yuly E. Sa´nchez1. 1 Physics, National University of Colombia, Bogota, Colombia, 2 Fundacio´n Instituto Inmunologico DE Colombia, Bogota, Colombia. The X-ray difraction and the nuclear magnetic recsonance (NMR) are the most comun methos for the determination of the tridimentional structure of peptides and proteins. The nmr allows to know the structure of the molecule simulating their natural environment, which does not allow X-ray difraction, it gives us a more accurate idea of the molecular estructure, because these structures show a dinamic due to the flexibility of the lateral chains. This paper aims show the process of determining the structure of a peptide residue of the Myelin basic protein (MBP) simulating their natural environment, through the use of NMR.