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Optimizing Protein Dynamics in Metalloenzyme Design
Monday, February 13, 2017 952-Pos Board B20 Dynamic Changes in Complement Component 3 in the Presence of the Lectin-Like Domain of Thrombomodulin Thomas Holt1, Daniel DeHelian1, Gavin Palowitch1, Julia R. Koeppe2. 1 Chemistry, Ursinus College, Collegeville, PA, USA, 2Chemistry, State University of New York at Oswego, Oswego, NY, USA. Component C3 is at the junction of three different complement activation pathways (classical, lectin, and alternative). Activation to C3b is a key step in the innate immune response that allows for the formation of important multiprotein complexes that ultimately participate in pathogen clearance. When misregulated, however, complement can lead to inflammatory disease and autoimmune disorders. Several regulatory proteins for C3b are known, but the molecular details of interactions between these proteins have not yet been elucidated. Thrombomodulin (TM), and specifically its N-terminal lectin-like domain (TMD1), has been identified as a possible regulator of complement through interactions with C3 or C3b. We have used fluorescence-based assays and hydrogen/deuterium exchange mass spectrometry (HDXMS) to study the interaction of TMD1 with C3 and C3b. We have found that TMD1 interacts with C3b, and there is a lesser interaction with C3. TMD1 tends to make C3b more accessible to deuterium exchange, while C3 tends to be less accessible to deuterium exchange in the presence of TMD1. This difference suggests a possible role for TMD1 in regulating a key step along the complement pathway. 953-Pos Board B21 Heat-Induced Dimerization of Colicin a Pore Forming Domain Yan Huang1, Jun-Peng Fan2, Jeremy H. Lakey3, Yi Liu1. 1 molecular and chemical school, Wuhan, China, 2Life sciences, Wuhan, China, 3cells and molecular biosciences, Newcastle upon tyne, United Kingdom. The differential scanning calorimetry (DSC) shows that the DHtr was always less than the DHvt’H (van’t Hoff) value and a the measured DHtr was always half the DHvt’H (van’t Hoff) value. And highly reproducible DHvt’H z 2 DHtr relationship could normally be explained if (a) the values of DHtr and DHvt’H are the same then the transition can be regarded as a two-state transition. (b) the cooperative unit is smaller than the macromolecule, as would be the case for a dimer, the DHtr is half of the DHvt’H. (c) in the case of a non-2-state processes, DHtr is larger than DHvt’H. The previously study shows that the ColA-P is monomer in solution at room temperature and low temperatures. The recent study demonstrated that although high energetic barrier between the dimer and monomer forms of ColA-P prevents them from interconverting at room temperature, the dimerization of ColA-P can be easily proceeded at high temperature. Dimerization can be observed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis at 50oC and confirmed by the highly reproducible DHvt’H z 2 DHtr relationship. 954-Pos Board B22 Effects of Humanizing Mutations on the Heme Crevice Loop of Yeast Iso1-Cytochrome c Haotian Lei, Bruce E. Bowler. Chemistry, University of Montana, Missoula, MT, USA. Cytochrome c (Cytc) is a small, globular protein within the inner mitochondrial membrane that plays an important role in cellular energy production by facilitating electron transport. Upon release from the mitochondria, Cytc participates in initiation of the caspase cascade leading to apoptosis, or programmed cell death. On the inner mitochondria membrane, Cytc binds cardiolipin, leading to loss of Met80-heme ligation, enabling Cytc to function as a peroxidase capable of oxidizing cardiolipin. Cytc has decreased affinity for oxidized cardiolipin and therefore can dissociate from the membrane facilitating release from mitochondrial and propagation of apoptosis. In order for the heme coordination site to open for peroxidase activity, the highly evolutionally-conserved heme crevice loop must undergo dynamic movement similar to the alkaline conformational transition. Unlike most residues in the heme crevice loop of Cytc, position 83 evolves to more sterically demanding residues in higher eukaryotes. We hypothesize that a more sterically demanding residue at position 83 would decrease the dynamics of the heme crevice loop and thus decrease peroxidase activity. By placing a valine at position 83 in yeast iso-1-cytochrome c, we are able to make the yeast Cytc heme crevice loop more human-like. Using UV-visible and circular dichroism spectroscopies, we have investigated global stability of the protein and local stability of the heme crevice loop. Using stopped-flow methods, we investigated the peroxidase activity for both G83V and E. coli expressed yWT(K72) yeast Cytc. We observe the surprising result that this mutation stabilizes the native conformer at alkaline pH, but destabilizes it at acidic pH. As a result, the G83V mutation causes higher than wild type peroxidase activity at acidic pH and lower than wild type peroxidase activity at alkaline pH.
193a
Protein Structure, Prediction, and Design II 955-Pos Board B23 Energy Landscape of a Bridge between Protein Folds Pengfei Tian, Robert B. Best. National Institute of Health, Bethesda, MD, USA. The entire structure space of the protein universe, so called fold-space, is estimated to contain thousands of different folds. Most proteins displaying high sequence identity are homologous and thus share the same fold, however, remarkable exceptions continue to be discovered. Some naturally occurring protein sequences can switch between different folds triggered by environmental changes. In addition, recent protein design experiments on two domains of different structure (all-alpha GA and alpha/beta GB from streptococcal protein G) have delineated a sequence of mutations linking two folds, suggesting how a new fold could arise from an existing one. This raises many interesting questions, including: How difficult is it to evolve from one fold to another? How many possible paths are there between folds and what are their common features? We have addressed such questions using a theoretical model of protein evolution, which builds on recent developments in statistical energy functions capturing co-evolutionary variation within a set of related proteins. We have applied the model to an experimentally well-characterized set of mutant sequences which bridge the fold landscape between GA and GB. It can capture both the trends in stability of the folded state within each fold, and also the propensity of the individual sequences to fold to a given structure. By using computer simulation to mimic the evolutionary dynamics, the mutational bridge between GA and GB in sequence space is characterized. Without a constraint to remain folded, the favored path between the two folds go via unfolded sequences. This suggests that the switch may occur via intrinsically disordered intermediate sequences which only fold on encountering their cognate ligands. However, there are still many accessible bridge sequences after a requirement of folded state stability is imposed. This model provides a new strategy of designing fold-switching proteins. 956-Pos Board B24 Optimizing Protein Dynamics in Metalloenzyme Design Ronald L. Koder, Bernard Everson, Lei Zhang, Jonathan Preston, Emma Bjerkefeldt. Physics, The City College of New York, New York, NY, USA. The control of dynamic motion in proteins is an underappreciated yet critical component in metalloprotein design. Our lab has performed the stepwise improvement of two designed enzymes - a heme based nitric oxide dioxygenase (NOD) and a dimanganese catalase - by significantly decreasing and increasing enzyme dynamics respectively. In the case of the artificial NOD, which was designed using our patterning-based inside-out algorithm, the protein is too dynamic, and we have extended the lifetime of the oxyferrous state intermediate 20-fold by dynamically restricting water access, thus decreasing futile cycling. The end result is a designed enzyme with a catalytic rate comparable to that of its natural counterparts. In the case of the catalase, which was designed using the DeGrado group’s physicsbased Pro-CAD computational design package, disruption of the wellpacked hydrophobic core increased the catalytic rate more than 10,000-fold over the starting enzyme. These results demonstrate that the low catalytic rates achieved in prior design efforts are a consequence of the failure of the designers to properly take into account the dynamic motions inherent to enzyme function. 957-Pos Board B25 A Statistical Mechanical Model for Coiled-Coil Protein Structure Prediction Mojtaba Jokar, Korosh Torabi. Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, USA. A coiled-coil protein structure consists of two or more interacting a-helical strands that together form a supercoil structure. Coiled-coil structures are critical to the function and integrity of various motor proteins, cytoskeletal filaments and extra-cellular matrix proteins. The ability to predict the propensity of two polypeptide strands to form a coiled-coil structure, at different solution conditions and external mechanical force is of special interest to a wide range of protein structure-function studies. Here we present a statistical mechanical model to predict the propensity of a given protein sequence to form a coiledcoil dimer. We calculate our model parameters based on experimental, structural and computational studies of a-helical and coiled-coil proteins. Further incorporation of this model into our previously developed a-helix tensile mechanics model enables us to predict the response of a coiled-coil structure to mechanical tension.
193a
Protein Structure, Prediction, and Design II 955-Pos Board B23 Energy Landscape of a Bridge between Protein Folds Pengfei Tian, Robert B. Best. National Institute of Health, Bethesda, MD, USA. The entire structure space of the protein universe, so called fold-space, is estimated to contain thousands of different folds. Most proteins displaying high sequence identity are homologous and thus share the same fold, however, remarkable exceptions continue to be discovered. Some naturally occurring protein sequences can switch between different folds triggered by environmental changes. In addition, recent protein design experiments on two domains of different structure (all-alpha GA and alpha/beta GB from streptococcal protein G) have delineated a sequence of mutations linking two folds, suggesting how a new fold could arise from an existing one. This raises many interesting questions, including: How difficult is it to evolve from one fold to another? How many possible paths are there between folds and what are their common features? We have addressed such questions using a theoretical model of protein evolution, which builds on recent developments in statistical energy functions capturing co-evolutionary variation within a set of related proteins. We have applied the model to an experimentally well-characterized set of mutant sequences which bridge the fold landscape between GA and GB. It can capture both the trends in stability of the folded state within each fold, and also the propensity of the individual sequences to fold to a given structure. By using computer simulation to mimic the evolutionary dynamics, the mutational bridge between GA and GB in sequence space is characterized. Without a constraint to remain folded, the favored path between the two folds go via unfolded sequences. This suggests that the switch may occur via intrinsically disordered intermediate sequences which only fold on encountering their cognate ligands. However, there are still many accessible bridge sequences after a requirement of folded state stability is imposed. This model provides a new strategy of designing fold-switching proteins. 956-Pos Board B24 Optimizing Protein Dynamics in Metalloenzyme Design Ronald L. Koder, Bernard Everson, Lei Zhang, Jonathan Preston, Emma Bjerkefeldt. Physics, The City College of New York, New York, NY, USA. The control of dynamic motion in proteins is an underappreciated yet critical component in metalloprotein design. Our lab has performed the stepwise improvement of two designed enzymes - a heme based nitric oxide dioxygenase (NOD) and a dimanganese catalase - by significantly decreasing and increasing enzyme dynamics respectively. In the case of the artificial NOD, which was designed using our patterning-based inside-out algorithm, the protein is too dynamic, and we have extended the lifetime of the oxyferrous state intermediate 20-fold by dynamically restricting water access, thus decreasing futile cycling. The end result is a designed enzyme with a catalytic rate comparable to that of its natural counterparts. In the case of the catalase, which was designed using the DeGrado group’s physicsbased Pro-CAD computational design package, disruption of the wellpacked hydrophobic core increased the catalytic rate more than 10,000-fold over the starting enzyme. These results demonstrate that the low catalytic rates achieved in prior design efforts are a consequence of the failure of the designers to properly take into account the dynamic motions inherent to enzyme function. 957-Pos Board B25 A Statistical Mechanical Model for Coiled-Coil Protein Structure Prediction Mojtaba Jokar, Korosh Torabi. Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, USA. A coiled-coil protein structure consists of two or more interacting a-helical strands that together form a supercoil structure. Coiled-coil structures are critical to the function and integrity of various motor proteins, cytoskeletal filaments and extra-cellular matrix proteins. The ability to predict the propensity of two polypeptide strands to form a coiled-coil structure, at different solution conditions and external mechanical force is of special interest to a wide range of protein structure-function studies. Here we present a statistical mechanical model to predict the propensity of a given protein sequence to form a coiledcoil dimer. We calculate our model parameters based on experimental, structural and computational studies of a-helical and coiled-coil proteins. Further incorporation of this model into our previously developed a-helix tensile mechanics model enables us to predict the response of a coiled-coil structure to mechanical tension.