Alternative splicing provides a mechanism for increasing function diversity in eukaryotic organisms. Splice variants (isoforms) are prevalent in the human genome. In the proposed study, the amino acid sequences of isoforms of human genes are mapped onto the three-dimensional structures of homologous proteins that share the same fold. The inferred structural modifications fall into at least six classes. Targets are selected for structural studies on the basis of functional effect of the alternative splicing, involvement of alternative splicing in disease, and the role of structure in mediating alternative function. We will determine the structures of at least 50 isoforms in the course of five years. The goal of the protein production component of the project is to supply sufficient amounts of highly purified protein samples for structural studies. For cases where the functions of the isoforms are still unknown, we will also supply clones and pure proteins to collaborators who will investigate their biological and biochemical functions. Because of the attrition when proceeding from genes to structures, work will be initiated on several hundreds of proteins. To achieve this goal, we will obtain cDNAs either from outside sources or by gene synthesis. To address the challenges associated with producing large amounts of soluble human proteins, we will develop and implement a flexible cloning system for exploring multiple expression vectors. The system is based on the high throughput cloning and expression platform developed during the current Program Project. We will vary the domain boundaries of the targeted genes, test for soluble protein expression, and engineer alternate splice versions. We will further expand the system to utilize fusion proteins, to explore in vitro folding from bacterial inclusion bodies, and to express soluble protein in eukaryotic systems. Soluble proteins will be purified by the most efficient protocols identified.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM057890-09
Application #
7553215
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
9
Fiscal Year
2006
Total Cost
$363,762
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
DUNS #
603819210
City
Baltimore
State
MD
Country
United States
Zip Code
21202
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Chen, Chen; Gorlatova, Natalia; Kelman, Zvi et al. (2011) Structures of p63 DNA binding domain in complexes with half-site and with spacer-containing full response elements. Proc Natl Acad Sci U S A 108:6456-61
Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin et al. (2010) Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member. Biochemistry 49:2031-41
Chen, Chen; Sun, Qihong; Narayanan, Buvaneswari et al. (2010) Structure of oxalacetate acetylhydrolase, a virulence factor of the chestnut blight fungus. J Biol Chem 285:26685-96
Melamud, Eugene; Moult, John (2009) Stochastic noise in splicing machinery. Nucleic Acids Res 37:4873-86
Melamud, Eugene; Moult, John (2009) Structural implication of splicing stochastics. Nucleic Acids Res 37:4862-72
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Chao, Kinlin L; Lim, Kap; Lehmann, Christopher et al. (2008) The Escherichia coli YdcF binds S-adenosyl-L-methionine and adopts an alpha/beta-fold characteristic of nucleotide-utilizing enzymes. Proteins 72:506-9
Zhuang, Zhihao; Song, Feng; Zhao, Hong et al. (2008) Divergence of function in the hot dog fold enzyme superfamily: the bacterial thioesterase YciA. Biochemistry 47:2789-96
Sari, Nese; He, Yanan; Doseeva, Victoria et al. (2007) Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae. Protein Sci 16:977-82

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