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STUDY OF STRUCTURAL FEATURES OF PROTEINS OF BIOTECHNOLOGICAL INTEREST BY MD SIMULATIONS Anna Marabotti Dept. Chemistry and Biology, University of Salerno, Fisciano (SA), Italy Molecular dynamics simulations are a powerful tool to investigate the structural features of proteins at atomic level, and in particular to introduce flexibility and temporal evolution in the analysis of molecular systems. During these years I have been involved in the study of many different proteins with potential application in biotechnology. The knowledge of their dynamics using a MD approach, coupled to the experimental evaluation of their structural and functional properties, has allowed to gain and integrate information at different levels, with a better comprehension of molecular phenomena associated to physico-chemical perturbations. Molecular dynamics simulations are a powerful tool to investigate the structural features of proteins at atomic level, and in particular to introduce flexibility and temporal evolution in the analysis of molecular systems. During these years I have been involved in the study of many different proteins with potential application in biotechnology. The knowledge of their dynamics using a MD approach, coupled to the experimental evaluation of their structural and functional properties, has allowed to gain and integrate information at different levels, with a better comprehension of molecular phenomena associated to physico-chemical perturbations. MD simulations have been performed using the open-source software GROMACS, running in parallel (MPI) on the CRESCO clusters CRESCO1 and 2. The length of simulation was variable between 10 and 100 ns. In general, simulations were run on 96 nodes, obtaining a performance of 15 ns/day (previous benchmarks performed showed that this combination is the best one in terms of scalability of the performances on these systems). At the end of simulations, several analyses were conducted using programs built within GROMACS, and results were visualized and elaborated with the aid of the freely available program Grace. MD simulations have been performed using the open-source software GROMACS, running in parallel (MPI) on the CRESCO clusters CRESCO1 and 2. The length of simulation was variable between 10 and 100 ns. In general, simulations were run on 96 nodes, obtaining a performance of 15 ns/day (previous benchmarks performed showed that this combination is the best one in terms of scalability of the performances on these systems). At the end of simulations, several analyses were conducted using programs built within GROMACS, and results were visualized and elaborated with the aid of the freely available program Grace. Arginine-binding protein (ArgBP) from T. maritima: analysis of its thermostability in different pH conditions. ArgBP, open form ArgBP, close form Bovine Odorant-binding protein (bOBP): analysis of its stability towards pressure stress MD simulations on this protein in different conditions of pH and temperature showed that helices appear to be more unstable than sheets to thermal stress (above). Three -strands (indicated with red boxes in the picture on the left), that are formed by hydrophobic residues and are completely shielded from solvent in the protein core, appear to be unaltered in any condition and seem to be essential for protein thermostability. Ref.: Scir A, Marabotti A, Staiano M, Iozzino L, Luchansky MS, Der BS, Dattelbaum JD, Tanfani F, D'Auria S. Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima. 2. Molecular organization and structural stability. Mol Biosyst. 2010;6(4):687-98. bOBP The MD simulations showed that, in native conditions, bOBP has a strong intrinsic resistance to high pressure (up to 600 bar) and keeps its dimeric assembly essentially unaltered, as shown by the conservation of the distance between center of mass of each monomer (picture on the middle). The comparison of the representative structures for each pressure (picture on the right) shows that the differences between them are focused mainly in the loops connecting the strands forming the central -barrel. Hence, dimerization and substrate binding significantly increase the resistance of the protein to pressure. Ref.: Marchal S, Marabotti A, Staiano M, Varriale A, Domaschke T, Lange R, D'Auria S. Under pressure that splits a family in two. The case of lipocalin family. PLoS One. 2012;7(11):e50489. Maltotriose binding protein (MalE2) from T. thermophilus: effect of pH on protein's fold MalE2 Ref.: Varriale A, Marabotti A, Mei G, Staiano M, D'Auria S. Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins. PLoS One 2013; 8(6):e64840. The MD simulations in different pH conditions showed that MalE2 at pH 4 is kinetically more unstable than in the other two pH conditions (7 and 10). The analysis of cluster structures (middle) showed that at pH 7 and 10 there is essentially a unique structure that prevails along the entire simulation, with multiple subpopulations in the case of higher pH. Instead, at acidic pH, MalE2 shows an enhanced instability of its tertiary structure. The representation of free energy landscape of the protein indicates the presence of multiple protein conformations at pH 4 and 10, probably due to the partial unfolding of the MalE2 tertiary structure, as a consequence of the induced perturbation of native ionic interactions.