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This thesis explores aspects protein function, structure and sequence by computational approaches. A comparative study of definitions of protein secondary structures was performed. Disagreements in assignment resulting from three different algorithms were observed. The causes of inaccuracies in structure assignments were discussed and possibilities of projecting protein secondary structures by different structural descriptors were tested. The investigation of inconsistent assignments of protein secondary structure led to a study of a more specific issue concerning protein structure/function relationships, namely cis/trans isomerisation of a peptide bond. Surveys were carried out at the level of protein molecules to detect the occurrences of the cis peptide bond, and at the level of protein domains to explore the possible biological implications of the occurrences of the structural motif. Research was then focussed on andalpha;-helical integral membrane proteins. A detailed analysis of sequences and putative transmembrane helical structures was conducted on the ABC transporters from different organisms. Interesting relationships between protein sequences, putative a-helical structures and transporter functions were identified. Applications of molecular dynamics simulations to the transmembrane helices of a specific human ABC transporter, cystic flbrosis transmembrane conductance regulator (CFTR), explored some of these relationships at the atomic resolution. Functional and structural implications of individual residues within membrane-spanning helices were revealed by these simulations studies.

Protein scaffolds are described as polypeptide frameworks with well-defined tertiary structures that are tolerable to mutagenesis or insertions. These scaffolds have gained significant interest from researchers and clinicians as they have challenged immunoglobulin domains as the preferred protein to address critical problems in biomedical engineering and biotechnology. While engineered antibodies and antibody fragments have been immensely successful, their complex structure, costly production and purification requirements, and large size preclude them from a host of applications. Small, stable proteins devoid of disulfide bond networks that express well recombinantly in prokaryotic systems offer viable alternatives to immunoglobulins. Repeat proteins are characterized structurally by tandem repeats of a consensus motif. These proteins are used in nature to mediate a variety of protein-protein interactions and are appealing scaffolds to bioengineers because of their predictable secondary structures. Several repeat scaffolds have been identified and successfully engineered for in vivo imaging and therapeutic applications. We have identified the repeats-in-toxin (RTX) protein as a potential antibody mimetic and interesting scaffold for protein engineering studies. RTX domains are commonly associated with extracellular proteins secreted through the type 1 secretion system in Gram-negative bacteria. They are composed of tandem repeats of a nonamer calcium binding sequence capped by N and C-termianl flanking regions. These proteins are conformationally dynamic and will fold from an intrinsically disordered state to a compact β-roll secondary structure in response to increasing calcium concentration. We aim to explore the RTX domain as an alternative protein scaffold and exploit the intrinsic conformational response to calcium as a mechanism to mediate molecular interactions. In our first study, we rationally engineer the RTX domain as a calcium-responsive physical cross-linker for hydrogel formation. Protein based materials are favorable for may biomedical applications because of their biocompatibility, tunable mechanical properties, and predictable erosion rates. We have designed a hydrophobic interface on the surface of the RTX domain that is present only in the calcium-bound β-roll conformation. In the absence of calcium, the peptide returns to its disordered state, delocalizing the hydrophobic patch and in turn mitigating the driving force for self-assembly. We show that these mutant RTX domains, with the aid of additional protein cross-linkers, self-assemble into cross-linked macromolecular hydrogel networks, only in the presence of calcium. To expand on this study, we further engineered the RTX domain to contain hydrophobic surfaces on both sides of the folded β-roll simultaneously. By doing this, we doubled the cross-linking capacity of the mutant RTX. This translates to a higher oligomerization state and lower protein concentration required for self-assembly. We also show the double mutant can function as a stand-alone cross-linking domain, eliminating the need for extraneous self-assembling proteins. This designed RTX mutant provides a new platform for stimulus-responsive cross-linking and self-assembly. In our next line of work, we created several synthetic RTX peptides based on a consensus design approach. Such an approach relies on identifying the minimal requirements for a single repeating unit, and concatenating the unit to achieve a desired protein interface. We identified the consensus nonameric unit for the RTX domain and generated several constructs of varying lengths using this sequence. However, it was discovered that these designed RTX peptides undergo a reversible phase change in response to calcium. Rather than abandon these synthetic peptides, we looked to use them as calcium-responsive protein purification tags. By appending a consensus RTX domain to a protein of interest, we were able rapidly and efficiently purify fusions out of cell lysate by precipitation cycling. We were also able to separate the tag from the protein of interest by including a protease recognition site between the two. This system offers an alternative to time consuming and expensive chromatographic techniques for recombinant protein purification. In our final study, we evaluated the RTX domain as a scaffold for evolving molecular recognition. We planned to use the calcium-responsive structural rearrangement as a switch to turn an evolved binding interface "on" and "off". One face of the folded β-roll structure was randomized on the genetic level and the resultant protein constructs were selected against a target protein using ribosome display technology. A consensus binding sequence emerged after several rounds of biopanning and was thoroughly characterized. The evolved β-roll bound the target protein with low micromolar affinity. Although this weak attraction was not suitable for efficiently capturing the target protein in a packed column application, this work provides a platform for evolving the RTX protein for molecular recognition. Several strategies are discussed to achieve higher affinity binders. Overall, this dissertation explores the RTX domain as an alternative stimulus-responsive scaffold for use in a variety of biotechnology applications. We have successfully developed new protein based platforms based on rationally designed or combinatorailly selected RTX proteins for calcium-responsive biomaterials, non-chromatographic protein purification, and calcium-dependent molecular recognition.

In recent years, the determination of several high-resolution structures of transient receptor potential (TRP) channels has led to significant progress within this field. The primary focus of this dissertation is to elucidate the structural characterization of TRPML1 and TRPP2. Mutations in TRPML1 cause mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We determined the first high-resolution crystal structures of the human TRPML1 I-II linker domain using X-ray crystallography at pH 4.5, pH 6.0, and pH 7.5. These structures revealed a tetramer with a highly electronegative central pore which plays a role in the dual Ca2+/pH regulation of TRPML1. Notably, these physiologically relevant structures of the I-II linker domain harbor three MLIV-causing mutations. Our findings suggest that these pathogenic mutations destabilize not only the tetrameric structure of the I-II linker, but also the overall architecture of full-length TRPML1. In addition, TRPML1 proteins containing MLIV-causing mutations mislocalized in the cell when imaged by confocal fluorescence microscopy. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD). Since novel technological advances in single-particle cryo-electron microscopy have now enabled the determination of high-resolution membrane protein structures, we set out to solve the structure of TRPP2 using this technique. Our investigations offer valuable insight into the optimization of TRPP2 protein purification and sample preparation procedures necessary for structural analysis.

An important problem in molecular biology is to predict the secondary structure of proteins from their primary structure. The primary structure of a protein is the sequence of amino acid residues. The secondary structure is an abstract description of the shape of the folded protein, with regions identified as alpha helix, beta strands, and random coil. Existing methods of secondary structure prediction examine a short segment of the primary structure and predict the secondary structure class (alpha, beta, coil) of an individual residue centered in that segment. The last few years of research have failed to improve these methods beyond the level of 65% correct predictions. This thesis investigates whether these methods can be improved by permitting them to examine externally-supplied predictions for the secondary structure of other residues in the segment. The externally-supplied predictions are called the "classification context," because they provide contextual information about the secondary structure classifications of neighboring residues. The classification context could be provided by an existing algorithm that made initial secondary structure predictions, and then these could be taken as input by a second algorithm that would attempt to improve the predictions. A series of experiments on both real and simulated classification context were performed to measure the possible improvement that could be obtained from classification context. The results showed that the classification context provided by current algorithms does not yield improved performance when used as input by those same algorithms. However, if the classification context is generated by randomly damaging the correct classifications, substantial performance improvements are possible. Even small amounts of randomly damaged correct context improves performance. / Graduation date: 1994

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published_or_final_version / abstract / toc / Electrical and Electronic Engineering / Master / Master of Philosophy

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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Barry, Bridette; Committee Member: Doyle, Donald; Committee Member: Kelly, Wendy; Committee Member: McCarty, Nael; Committee Member: Schimdt-Krey, Ingaborg. Part of the SMARTech Electronic Thesis and Dissertation Collection.