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Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin
Sunday, February 12, 2017 domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH. We find that acidification of the cytoplasm results in increased PI(4,5)P2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift. In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs. This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O. 339-Pos Board B104 Electric Field Effects in the Active Site of a Thermophilic Enzyme as Observed by FTIR and 2D IR Spectroscopy Tayler D. Hill, Hannah H. Lepird, David A. Price, Sean D. Moran. Chemistry and Biochemistry, Southern Illinois University Carbondale, Carbondale, IL, USA. Our research aims to understand how changes in ultrafast dynamics compare and correlate to thermophilic enzyme activity. We observe fluctuating electric field effects in a promiscuous, hyperthermophilic ene-reductase from Pyrococcus horikoshii (PhENR) to address this. This enzyme catalyzes the reduction of activated alkenes/alkynes to their respective alkanes/alkenes via proton and hydride transfers from a flavin cofactor in the active site. We exploit the promiscuity of PhENR in order to incorporate a variety of substrates and substrate analogs into the active site for these studies. We have synthesized a set of covalently-attached substituted N-phenylmaleimide infrared labels, which mimic the structures of the enzyme’s substrates, and contain unique vibrational chromophores to probe the enzyme’s active site dynamics. Current studies focus on the vibrational frequencies and lineshapes of nitrile labels such as those of 4-cyano-N-phenylmaleimide, which sits proximal to the catalytic flavin and can be attached in multiple orientations within the active site. When compared to the label in solution, the covalently attached label undergoes significant inhomogeneous broadening in its FTIR spectrum reflecting the distribution of active site microenvironments. Additionally, protein-based non-natural amino acid labels such as methionine to azidohomoalanine substitutions are also being incorporated into the distal side of the flavin cofactor for similar studies in different location within the enzyme’s active site. Using 2D IR spectroscopy, we are examining the contributions of femtosecond to picosecond active site dynamics to the lineshapes of both the covalently attached probes as well as the incorporated non-natural amino acid labels. Future research aims to break the thermophilicity of the enzyme via specific mutations in order to compare the active site dynamics to a corresponding mesophilic version of the protein. 340-Pos Board B105 Infrared Structural Biology: How to Detect Protonation States of Histidine Side Chains in Proteins Aihua Xie, Charle Liu, Matthew Cavener. Physics, Oklahoma State University, Stillwater, OK, USA. The imidazole group of histidine residues are found functionally important in a vast number of catalytic proteins. The remarkable catalytic power of histidine side chains originates from its ionizable imidazole ring armed with a pair of different tertiary amines and the ability of adopting three protonation states near physiological pH environment. Knowledge on the protonation states of key histidine side chains in enzymes at rest and during catalytic actions is indispensable to elucidation of the structure-function relationship underlying enzymatic catalysis. We report a rigorous method on how to detect the three protonation states of functionally important histidine imidazole rings in the static and dynamic states of enzymes using infrared structural biology. First principle computational methods based on density functional theory were employed to develop two vibrational structural markers (VSM) of the imidazole group: VSMq for the charged states of the imidazole group, while VSMt for distinguishing the D and E tautomers of charge neutral histidine. The accuracy of the VSMs is assessed by comparison of calculated VSMs with experimental FT-IR data of the 4-ethyl-imidazole model compound. We will discuss how these VSMs may be employed in
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structure-function studies on functionally important histidine residues in enzymes. 341-Pos Board B106 Origin of Chain Length Specificites of Starch Branching Enzyme Hadi Nayebi Gavgani, Remie Fawaz, Zahra Assar, Alireza Ghanbarpour, David Walls, Sarah McGovern, James H. Geiger. Chemistry, Michigan State University, East Lansing, MI, USA. Starch Branching enzyme (BE) is one of the three enzymes involved in starch biosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches, remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. There are at least two isoforms of the enzyme in most plants, with each having distinct reactivities and product chain-length specificities. Though the chemistry of the active site of branching enzyme is relatively well studied, the origin of the chain length specificity is yet to be understood. Using protein crystallography and biochemical studies on rice branching enzyme, we aim to understand the factors controlling the chain-length specificity of branching enzymes. The mechanism of branching enzyme involves two steps: (1) An oligosaccharide (donor chain) binds to the enzyme and is cleaved by the action of a nucleophilic aspartate residue to form a covalently-linked enzyme-glucan intermediate. (2) A second oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of one of its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We have focused on discovering the surface glucan binding sites in branching enzyme because they are likely essential to understanding the specificity of the enzyme. To this end we have obtained a crystal structure of an oligosaccharide (M12)bound rice branching enzyme, which reveals oligosaccharide binding from the outer surface of the enzyme almost to the active site. Mutations of the residues interacting with the M12 can substantially compromise the enzyme’s activity, though none have affected the branch chain specificity. On the other hand, comparison of an isoamylase maltoheptaose (M7)-bound structure with rice branching enzyme suggested another potential glucan surface binding site. Interestingly, mutations in this new site did effect the chain-length specificity of the enzyme. These results suggest that the bound M12 is part of the acceptor chain and the newly identified binding site hosts the donor chain. Together, the data allow us to, for the first time propose a detailed mechanism for the enzyme, explaining how disparate surface glucan binding sites far from the active site create the enzyme’s activity and specificity against its polymeric substrate. 342-Pos Board B107 Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin Madison Tyler1, Corey Reed1, Dan Sackett2, Jennifer Ross1. 1 Physics, University of Massachusetts, Amherst, Amherst, MA, USA, 2 National Institutes of Health, Bethesda, MD, USA. Microtubules are part of a dynamic cytoskeletal network that is constantly being reorganized to control cell processes such as neuronal development and maintenance, cell division, and cargo transport. Many stabilizing and destabilizing enzymes function to reorganize these networks for the specific needs of the cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAAþ) family. It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules. Our lab has previously shown free tubulin dimers and CTTs alone can inhibit katanin severing. We seek to determine the manner that tubulin CTTs sequence can regulate katanin activity using polypeptide sequences of CTTs of different tubulin isoforms. We find that the sequence’s ionic, hydrophobic, and steric features play a role in determining katanin’s activity.
Ribosomes and Translation 343-Pos Board B108 Protein Synthesis Times Scale with Gene Length because the Determinants of Translation Speed are Randomly Distributed Across Genes Edward P. O’Brien, Ajeet Sharma. Department of Chemistry, Penn State University, University Park, PA, USA. Many of the molecular factors influencing codon translation speed have been identified, and their relative contributions estimated. These factors include tRNA concentration, the presence of charged nascent-chain residues in the ribosome exit tunnel, mRNA secondary structure, proline residues at the A or P sites of the ribosome and steric interactions between ribosomes translating a transcript. Here, we combine this information with genomic information from E. coli, yeast and humans in a simulation model of translation to estimate the synthesis time of cytopolasmic proteins. We find that regardless of the organism, the synthesis time of a protein scales linearly with the length of mRNA’s coding sequence even though there is a large variation in the translation speed of individual codons. We demonstrate that this scaling arises because the
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structure-function studies on functionally important histidine residues in enzymes. 341-Pos Board B106 Origin of Chain Length Specificites of Starch Branching Enzyme Hadi Nayebi Gavgani, Remie Fawaz, Zahra Assar, Alireza Ghanbarpour, David Walls, Sarah McGovern, James H. Geiger. Chemistry, Michigan State University, East Lansing, MI, USA. Starch Branching enzyme (BE) is one of the three enzymes involved in starch biosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches, remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. There are at least two isoforms of the enzyme in most plants, with each having distinct reactivities and product chain-length specificities. Though the chemistry of the active site of branching enzyme is relatively well studied, the origin of the chain length specificity is yet to be understood. Using protein crystallography and biochemical studies on rice branching enzyme, we aim to understand the factors controlling the chain-length specificity of branching enzymes. The mechanism of branching enzyme involves two steps: (1) An oligosaccharide (donor chain) binds to the enzyme and is cleaved by the action of a nucleophilic aspartate residue to form a covalently-linked enzyme-glucan intermediate. (2) A second oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of one of its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We have focused on discovering the surface glucan binding sites in branching enzyme because they are likely essential to understanding the specificity of the enzyme. To this end we have obtained a crystal structure of an oligosaccharide (M12)bound rice branching enzyme, which reveals oligosaccharide binding from the outer surface of the enzyme almost to the active site. Mutations of the residues interacting with the M12 can substantially compromise the enzyme’s activity, though none have affected the branch chain specificity. On the other hand, comparison of an isoamylase maltoheptaose (M7)-bound structure with rice branching enzyme suggested another potential glucan surface binding site. Interestingly, mutations in this new site did effect the chain-length specificity of the enzyme. These results suggest that the bound M12 is part of the acceptor chain and the newly identified binding site hosts the donor chain. Together, the data allow us to, for the first time propose a detailed mechanism for the enzyme, explaining how disparate surface glucan binding sites far from the active site create the enzyme’s activity and specificity against its polymeric substrate. 342-Pos Board B107 Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin Madison Tyler1, Corey Reed1, Dan Sackett2, Jennifer Ross1. 1 Physics, University of Massachusetts, Amherst, Amherst, MA, USA, 2 National Institutes of Health, Bethesda, MD, USA. Microtubules are part of a dynamic cytoskeletal network that is constantly being reorganized to control cell processes such as neuronal development and maintenance, cell division, and cargo transport. Many stabilizing and destabilizing enzymes function to reorganize these networks for the specific needs of the cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAAþ) family. It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules. Our lab has previously shown free tubulin dimers and CTTs alone can inhibit katanin severing. We seek to determine the manner that tubulin CTTs sequence can regulate katanin activity using polypeptide sequences of CTTs of different tubulin isoforms. We find that the sequence’s ionic, hydrophobic, and steric features play a role in determining katanin’s activity.
Ribosomes and Translation 343-Pos Board B108 Protein Synthesis Times Scale with Gene Length because the Determinants of Translation Speed are Randomly Distributed Across Genes Edward P. O’Brien, Ajeet Sharma. Department of Chemistry, Penn State University, University Park, PA, USA. Many of the molecular factors influencing codon translation speed have been identified, and their relative contributions estimated. These factors include tRNA concentration, the presence of charged nascent-chain residues in the ribosome exit tunnel, mRNA secondary structure, proline residues at the A or P sites of the ribosome and steric interactions between ribosomes translating a transcript. Here, we combine this information with genomic information from E. coli, yeast and humans in a simulation model of translation to estimate the synthesis time of cytopolasmic proteins. We find that regardless of the organism, the synthesis time of a protein scales linearly with the length of mRNA’s coding sequence even though there is a large variation in the translation speed of individual codons. We demonstrate that this scaling arises because the