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Hydrogen-Deuterium Exchange Mass Spectroscopy to Determine Structure and Structural Dynamics of Protein Complexes
Wednesday, February 15, 2017 paramagnetic resonance (EPR) oximetry have provided valuable estimates of membrane oxygen permeability. Yet, the estimates incorporate a good deal of uncertainty due to ambiguity in probe positioning. We use atomistic molecular dynamics simulations to examine the behavior of a probe targeted to the headgroup region of zwitterionic phospholipid bilayers. Our simulations are validated by detailed comparison with experimental data, including overall agreement with the EPR oximetry measurements. The simulations predict that the tempocholine ‘‘headgroup’’ probe oversamples the hydrophobic tail region of the bilayer, leading to an underestimate of resistance to permeation in the headgroup region and an overestimate of bilayer permeability. This work highlights the value of using atomistic simulations to test assumptions made in interpreting probe-based experimental measurements.
Platform: Protein Assemblies 2306-Plat In Situ Mechanical Interrogation of Single Nuclear Lamins Suggests the Lamina is a Robust Framework Tanuj Sapra1, Zhao Qin2, Markus Buehler2, Ohad Medalia1. 1 University of Zurich, Zurich, Switzerland, 2MIT, Cambridge, MA, USA. The nuclear lamina is a major structural element of the nucleus, predominately composed of the intermediate filament lamin proteins, enveloped by the nuclear membrane. Underlining its importance to the mechanical and structural integrity of the nucleus, a persistent goal has been to examine the lamin network in situ. Here we apply an integrative approach combining techniques from mechanobiology (atomic force microscopy), visual proteomics (cryo-electron tomography) and network analysis (molecular dynamic simulations) to understand the design principle of the lamin network. A detailed analysis of the mechanical failure under nano-Newton forces of the endogenous lamins of Xenopus laevis oocytes revealed the lamin filaments to be strong, stiff and tough, equivalent to natural silk and the synthetic polymer KevlarÒ. The combined approach is unique, and provides understanding of the structure-function of proteins involved in diseases from a materials science perspective, the underlying goal of ‘materiomics’. 2307-Plat Nano-Space Video Imaging Reveals Structural Dynamics of Fibrous Protein Assembly and Relevant Enzymes Takahiro Watanabe-Nakayama1, Noriyuki Kodera1, Hiroki Konno1, Kenjiro Ono2, David B. Teplow3, Masahito Yamada1, Toshio Ando1. 1 Kanazawa University, Kanazawa, Japan, 2Showa University, Tokyo, Japan, 3 UCLA, Los Angels, CA, USA. Protein assembly into fibrous structure is ubiquitous in biological systems. However, structural dynamics in assembling processes of these structures and mechanisms of relevant enzymatic reactions still have remained unclear due to insolubility of the high order structures. In this presentation, we report two application results of high-speed atomic force microscopy (HS-AFM) observation of such systems. HS-AFM visualize structural dynamics of all objects in an observation field regardless of their solubility. Bacterial collagenases are widely applied in many fields due to their high activity and specificity; however, little is known about the mechanisms by which bacterial collagenases degrade insoluble collagen. HS-AFM visualized ColG moved ~14.5 nm towards the collagen N terminus over ~3.8 s in a manner depending on a catalytic zinc ion, suggesting that ColG couples its movement and function with high efficiency. In addition, HS-AFM demonstrated ColG engagements were hindered by the hierarchical structure of collagen. Amyloid b-protein (Ab) aggregation into amyloid fibrils has been implicated in the pathogenesis of Alzheimer’s disease. Fibril formation is a complex process, but HS-AFM enables such observation. HS-AFM visualized two different growth modes of the 42-residue form of the amyloid b-protein (Ab1-42), one producing straight fibrils and the other producing spiral fibrils. Each mode depended on initial nucleus structure, but conformational switching was observed sometimes. The frequency of this switching phenomenon was varied in response to changes in micro environment around fibril ends. Our data provide the new insights into the template-dependent mechanism of Ab1-42 fibril formation. References T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils. Sci. Rep. 6:28975 (2016). T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals structural dynamics of amyloid b1-42 aggregates. Proc. Natl. Acad. Sci. U. S. A. 113(21):5835-40 (2016).
469a
2308-Plat Hydrophobic Interfaces, Key Regions for Assembly of Transmembrane Proteins : A Study of E. Coli Aquaporin Z Victoria Schmidt, James N. Sturgis. CNRS and Aix-Marseille univ., Marseille, France. Stable folding of membrane proteins depends on the formation of stable interfaces in the hydrophobic core of the membrane, and the exclusion of lipid molecules. There is very little information on this aspect of membrane protein folding for polytopic membrane proteins. We use Aquaporin Z as a model to study the structure and importance of these interfaces. Aquaporins are membrane proteins with 6 transmembrane helices, that act as channels in biological membranes for small uncharged molecules. Members of this family of proteins assemble in the membrane to form tetrameric complexes. In order to better understand the assembly of these proteins we have constructed a series of destabilized mutants by modifying the interface between monomers in Aquaporin Z from E. coli. We have characterized these mutants to understand how these modifications modulate in vivo and in vitro characteristics of the protein. In vivo we have evaluated membrane insertion and the formation of tetramers and toxicity. In vitro we have evaluated the tertiary and quaternary structure, stability and activity of the protein in detergents or lipid bilayers. 2309-Plat Hydrogen-Deuterium Exchange Mass Spectroscopy to Determine Structure and Structural Dynamics of Protein Complexes Emanuele Paci. University of Leeds, Leeds, United Kingdom. Hydrogen deuterium mass spectrometry (HDX-MS) is a growing technique to probe structure and dynamics of proteins and protein-ligand complexes. It is particularly powerful to probe disordered or partly disordered states, when no high-resolution structural techniques are applicable. It is now increasingly used as a fast and inexpensive approach to map how protein and ligands bind when structures are not available or when dynamical changes and allostery is involved. HDX-MS provides, as a function of time, the change in mass of random segments of the polypeptide chain where deuterated amide protons exchange with solvent’s hydrogens or vice-versa. The main drawback of the technique is that the information provided is intrinsically low-resolution and it is not directly interpretable in structural terms, the main reason being that exchange depends simultaneously on local structure and dynamics. We have developed a technique that allows accurate estimation of the exchange kinetics for any segment of the polypeptide chain based from ensembles of structures. The technique we developed builds up on the observation that the rate of exchange of a single amide proton (or equivalently, its protection factor) can be estimated from the protein structure by estimating the accessibility to the solvent and the probability of the amide being involved in a hydrogen bond. I will present published and unpublished results that demonstrate the potential of the technique to study the functional dynamics of a hexameric helicase [1] and to assess the quality of a model for a protein complex. 1. Radou, G., et al., Functional dynamics of hexameric helicase probed by hydrogen exchange and simulation. Biophys J, 2014. 107(4): p. 983-90. 2310-Plat Molecular Coevolution of Fli Proteins Provides a Guide to Accurate Models of Flagellar Protein Complexes and Dynamics Faruck Morcos. Biological Sciences, University of Texas at Dallas, Richardson, TX, USA. Elucidating the molecular mechanics of bacterial flagellar motor architecture has been a challenging topic for many years. Efforts in structural biochemistry, crystallography, electron microscopy and computational biology brought an improved picture of motor ring assemblies and dynamics. There are, however, many challenges to be resolved to fully understand the formation of this multiprotein macro complex. There are only a few experimentally determined 3D coordinates of the flagellar motor switch proteins. Proteins FliF, FliG, FliM and FliN constitute the rotor of the flagellar motor as well as scaffold for the Type III ATPase secretion complex responsible for assembly of external flagellar structures. Ring formation has been studied at lower resolutions compared to the individual monomers leaving open questions about the complex connectivity and the dynamics of switching and rotation. Some remaining questions are related to the nature of oligomerization and dimerization. We have studied the amino acid coevolution of Fli proteins, particularly FliM (middle and C-terminal domains) as well as protein FliN to elucidate important interactions conserved through evolution. We combine such signals with molecular dynamics to accurately recapitulate previous known complexes involving FliN homodimers as well as to provide support for some putative models of FliM-FliN dimers as well as distinct signatures for tetrametic
Platform: Protein Assemblies 2306-Plat In Situ Mechanical Interrogation of Single Nuclear Lamins Suggests the Lamina is a Robust Framework Tanuj Sapra1, Zhao Qin2, Markus Buehler2, Ohad Medalia1. 1 University of Zurich, Zurich, Switzerland, 2MIT, Cambridge, MA, USA. The nuclear lamina is a major structural element of the nucleus, predominately composed of the intermediate filament lamin proteins, enveloped by the nuclear membrane. Underlining its importance to the mechanical and structural integrity of the nucleus, a persistent goal has been to examine the lamin network in situ. Here we apply an integrative approach combining techniques from mechanobiology (atomic force microscopy), visual proteomics (cryo-electron tomography) and network analysis (molecular dynamic simulations) to understand the design principle of the lamin network. A detailed analysis of the mechanical failure under nano-Newton forces of the endogenous lamins of Xenopus laevis oocytes revealed the lamin filaments to be strong, stiff and tough, equivalent to natural silk and the synthetic polymer KevlarÒ. The combined approach is unique, and provides understanding of the structure-function of proteins involved in diseases from a materials science perspective, the underlying goal of ‘materiomics’. 2307-Plat Nano-Space Video Imaging Reveals Structural Dynamics of Fibrous Protein Assembly and Relevant Enzymes Takahiro Watanabe-Nakayama1, Noriyuki Kodera1, Hiroki Konno1, Kenjiro Ono2, David B. Teplow3, Masahito Yamada1, Toshio Ando1. 1 Kanazawa University, Kanazawa, Japan, 2Showa University, Tokyo, Japan, 3 UCLA, Los Angels, CA, USA. Protein assembly into fibrous structure is ubiquitous in biological systems. However, structural dynamics in assembling processes of these structures and mechanisms of relevant enzymatic reactions still have remained unclear due to insolubility of the high order structures. In this presentation, we report two application results of high-speed atomic force microscopy (HS-AFM) observation of such systems. HS-AFM visualize structural dynamics of all objects in an observation field regardless of their solubility. Bacterial collagenases are widely applied in many fields due to their high activity and specificity; however, little is known about the mechanisms by which bacterial collagenases degrade insoluble collagen. HS-AFM visualized ColG moved ~14.5 nm towards the collagen N terminus over ~3.8 s in a manner depending on a catalytic zinc ion, suggesting that ColG couples its movement and function with high efficiency. In addition, HS-AFM demonstrated ColG engagements were hindered by the hierarchical structure of collagen. Amyloid b-protein (Ab) aggregation into amyloid fibrils has been implicated in the pathogenesis of Alzheimer’s disease. Fibril formation is a complex process, but HS-AFM enables such observation. HS-AFM visualized two different growth modes of the 42-residue form of the amyloid b-protein (Ab1-42), one producing straight fibrils and the other producing spiral fibrils. Each mode depended on initial nucleus structure, but conformational switching was observed sometimes. The frequency of this switching phenomenon was varied in response to changes in micro environment around fibril ends. Our data provide the new insights into the template-dependent mechanism of Ab1-42 fibril formation. References T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils. Sci. Rep. 6:28975 (2016). T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals structural dynamics of amyloid b1-42 aggregates. Proc. Natl. Acad. Sci. U. S. A. 113(21):5835-40 (2016).
469a
2308-Plat Hydrophobic Interfaces, Key Regions for Assembly of Transmembrane Proteins : A Study of E. Coli Aquaporin Z Victoria Schmidt, James N. Sturgis. CNRS and Aix-Marseille univ., Marseille, France. Stable folding of membrane proteins depends on the formation of stable interfaces in the hydrophobic core of the membrane, and the exclusion of lipid molecules. There is very little information on this aspect of membrane protein folding for polytopic membrane proteins. We use Aquaporin Z as a model to study the structure and importance of these interfaces. Aquaporins are membrane proteins with 6 transmembrane helices, that act as channels in biological membranes for small uncharged molecules. Members of this family of proteins assemble in the membrane to form tetrameric complexes. In order to better understand the assembly of these proteins we have constructed a series of destabilized mutants by modifying the interface between monomers in Aquaporin Z from E. coli. We have characterized these mutants to understand how these modifications modulate in vivo and in vitro characteristics of the protein. In vivo we have evaluated membrane insertion and the formation of tetramers and toxicity. In vitro we have evaluated the tertiary and quaternary structure, stability and activity of the protein in detergents or lipid bilayers. 2309-Plat Hydrogen-Deuterium Exchange Mass Spectroscopy to Determine Structure and Structural Dynamics of Protein Complexes Emanuele Paci. University of Leeds, Leeds, United Kingdom. Hydrogen deuterium mass spectrometry (HDX-MS) is a growing technique to probe structure and dynamics of proteins and protein-ligand complexes. It is particularly powerful to probe disordered or partly disordered states, when no high-resolution structural techniques are applicable. It is now increasingly used as a fast and inexpensive approach to map how protein and ligands bind when structures are not available or when dynamical changes and allostery is involved. HDX-MS provides, as a function of time, the change in mass of random segments of the polypeptide chain where deuterated amide protons exchange with solvent’s hydrogens or vice-versa. The main drawback of the technique is that the information provided is intrinsically low-resolution and it is not directly interpretable in structural terms, the main reason being that exchange depends simultaneously on local structure and dynamics. We have developed a technique that allows accurate estimation of the exchange kinetics for any segment of the polypeptide chain based from ensembles of structures. The technique we developed builds up on the observation that the rate of exchange of a single amide proton (or equivalently, its protection factor) can be estimated from the protein structure by estimating the accessibility to the solvent and the probability of the amide being involved in a hydrogen bond. I will present published and unpublished results that demonstrate the potential of the technique to study the functional dynamics of a hexameric helicase [1] and to assess the quality of a model for a protein complex. 1. Radou, G., et al., Functional dynamics of hexameric helicase probed by hydrogen exchange and simulation. Biophys J, 2014. 107(4): p. 983-90. 2310-Plat Molecular Coevolution of Fli Proteins Provides a Guide to Accurate Models of Flagellar Protein Complexes and Dynamics Faruck Morcos. Biological Sciences, University of Texas at Dallas, Richardson, TX, USA. Elucidating the molecular mechanics of bacterial flagellar motor architecture has been a challenging topic for many years. Efforts in structural biochemistry, crystallography, electron microscopy and computational biology brought an improved picture of motor ring assemblies and dynamics. There are, however, many challenges to be resolved to fully understand the formation of this multiprotein macro complex. There are only a few experimentally determined 3D coordinates of the flagellar motor switch proteins. Proteins FliF, FliG, FliM and FliN constitute the rotor of the flagellar motor as well as scaffold for the Type III ATPase secretion complex responsible for assembly of external flagellar structures. Ring formation has been studied at lower resolutions compared to the individual monomers leaving open questions about the complex connectivity and the dynamics of switching and rotation. Some remaining questions are related to the nature of oligomerization and dimerization. We have studied the amino acid coevolution of Fli proteins, particularly FliM (middle and C-terminal domains) as well as protein FliN to elucidate important interactions conserved through evolution. We combine such signals with molecular dynamics to accurately recapitulate previous known complexes involving FliN homodimers as well as to provide support for some putative models of FliM-FliN dimers as well as distinct signatures for tetrametic