Life Sciences Commons^™

Expression, Purification, And Analysis Of Unknown Translation Factors From Escherichia Coli: A Synthesis Approach, Justin D. Walter, Peter Littlefield, Scott P. Delbecq, Gerry Prody, P. Clint Spiegel

Chemistry Faculty and Staff Publications

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity …

The Tertiary Structure And Domain Organization Of Coagulation Factor Viii, P. Clint Spiegel, Betty W. Shen, Chong-Hwan Chang, Jae-Wook Huh, Jeanman Kim, Young-Ho Kim, Barry L. Stoddard

Chemistry Faculty and Staff Publications

Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 do- mains. The structure permits unambiguous modeling of the relative orientations of the 5 domains of r-fVIII. …

Elongation Factor G Stabilizes The Hybrid-State Conformation Of The 70s Ribosome, P. Clint Spiegel, Dmitri N. Ermolenko, Harry F. Noller

Chemistry Faculty and Staff Publications

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP·fusidic acid induces movement of a deacylated tRNA from …

Structural And Biochemical Analyses Of Dna And Rna Binding By A Bifunctional Homing Endonuclease And Group I Intron Splicing Factor, Jill M. Bolduc, P. Clint Spiegel, Pivali Chatterjee, Kristina L. Brady, Maureen E. Downing, Mark G. Caprara, Richard B. Waring, Barry L. Stoddard

Chemistry Faculty and Staff Publications

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 Å resolution. The structure demonstrates the remarkable structural conservation of the (3-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant …