Analyzing the Molecular-level Roles and Effects of Small Molecule Inclusions in Crytallization Experiments Involving Lysozyme and DmxA, Holmes, JB
Institute of Nanobiology and Structural Biology, Academic and University Ceter Nové Hrady / University of South Bohemia Institute of Physical Biology, Academy of Sciences of the Czech Republic Institute of Systems Biology and Ecology
Professor: Dr. Ivana Kutá Smatanova
Crystallization of biomacromolecules is a nontrivial task and represents a major bottleneck in solving the three-dimensional structure by means of x-ray diffraction of new and unique biopolymers and their complexes, in studying and understanding enzymatic or structural function of some of the most biologically and medically significant targets, and in the rational design of structure-based pharmaceuticals. Macromolecular crystals generally suffer by having poor mechanical and optical properties, nonetheless, depending on the size and quality/strength of the crystal lattice may be used as diffraction grating for interacting with x-rays and generating useful patterns of deflections from which the three-dimensional structure of biological architecture is determinable at atomic resolutions. Biomacromolecular crystals are grown in aqueous solutions called mother liquors and is based on molecular recognition resulting from extensive natural selection/evolution of protein biochemistry. At increasing particle density, specific chemical attraction results in the onset of macrobonds on mutually compatible surfaces between protein molecules within the crystal. Crystals form in non-equilibrium or supersaturated solutions as protein monomers are slowly precipitated out of solution in the insoluble form in such way as a highly ordered solid crystalline state is formed. Non-equilibrium thermodynamics drives the nucleation and growth kinetics of crystal formation and represents a first-order phase transformation in the state of matter. Partition of protein into two distinct phases, the liquid mother phase and the solid crystal phase, may be theoretically considered by two dimensional or greater dimensional phase diagrams that describes the state of a material as a function of the ambient conditions (i.e. pH or precipitant concentration). Typically the individual solution components of the mother liquor consists of a suitable buffer, organic and/or inorganic salts, polymeric compounds such as PEG, and a broad range of organic and inorganic small molecule additives are also included in the solution chemistries. Many examples from past experiments involving additives has demonstrated their usefulness in increasing the probabilities and reproducibility of crystallization trials and show that often inclusions are based on no apparent rationale but essential in the crystallization process of some intractable targets such as membrane proteins and glycoproteins. In the research presented here, in total, 96 individual small molecule additives over a broad range of chemical classifications were assessed for their possible usefulness and to assess the possible applications lysozyme crystallization using additives as an early model to studying the molecular level role of additives . Crystal effects, defects, and statistics from experiments involving lysozyme and the future applications for future experiments in crystallogenesis will also be of discussion.
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