Life Sciences Commons^™

Blood Coagulation Factor Ix: Purification, Activation, Crystallization, Juliet Mcgill

WWU Honors College Senior Projects

This paper presents readers with an optimized procedure for the purification, activation, and crystallization of selected blood coagulation Factor IX double mutant (FIX_2). Through the completion of this work, we aim to enhance future biochemical and structural studies by providing an easier means for the FIX_2 production, in order to increase understanding of the protein’s function within the blood coagulation cascade. The initiation of the blood coagulation cascade is brought on by activation of inactive Factor VIII (FVIII) protein though contact with tissue factor, the FVIII protein then binds to an activated platelet surface where it must wait for its …

A Functional Study Of Plant Protein Villin4 And The Application Of Sortase Mediated Ligation In Intrinsically Disordered Protein Substrates, Jake Heins

WWU Graduate School Collection

Villin proteins are a family of filamentous actin (F-actin) regulators, playing vital roles in the organization of cytoskeletons in eukaryotes. Villin4, found in A. thaliana has a distinct structure that contains a folded C-terminal headpiece, a 192-residue disordered “linker”, and a folded N-terminal core domain. Villin4 has been shown to bind and bundle F-actin in the C-terminal headpiece domain. This disordered linker, or intrinsically disordered region (IDR), lacks the defined three-dimensional structure often found in proteins. IDRs are not novel. In fact, over 30% of eukaryotic proteins contain a sizable intrinsically disordered region over 50 amino acids in length. These …

Blood Coagulation Factor Ix: Purification, Isolation, Activation, Alex Macneil

WWU Honors College Senior Projects

This paper attempts to provide an optimized strategy for the purification, activation, and isolation of blood coagulation Factor IX mutants. The goal of this work is to enable future biochemical and structural studies of Factor IX to a gain a better understanding of the structural-functional role this protein plays in the blood coagulation cascade. The orchestration and amplification of the blood coagulation cascade requires the binding of Factor VIII (FVIII) to an activated platelet surface, where it serves as a cofactor to a serine protease, Factor IX (FIX). Factor IX circulates the bloodstream as a catalytically silent multidomain protein1. Like …

Structural Analysis Of Protein-Peptide Interactions, Melody Gao

WWU Honors College Senior Projects

Over the last three years in the Amacher lab, I have been fortunate to work on two amazing projects studying protein-peptide interactions: PDZ domains and Class A sortases. Both recognize a certain substrate motif, and we are interested in these proteins' selectivity and promiscuity of their substrate.

Probing Large Intrinsically Disordered Regions Through Novel Sortase-Mediated Ligation, Leah Kjormoe

Scholars Week

In the realm of proteins, it is widely accepted that structure informs function. However, there are many proteins that contain intrinsically disordered regions (IDRs). These regions are areas in which the protein lacks defined structure, and IDPs are also often unstable, which complicates structural studies. NMR spectroscopy is an established method for probing protein structure and has been applied to that end in small IDRs. However, larger IDRs often have spectral overlap that makes data difficult to interpret. Furthermore, low-concentration samples limit spectral clarity. One method to address these difficulties is to use sortase ligation and segmental labeling, which increases …

Structure Determination Of A Bioengineered Human/Porcine Factor Viii For Hemophilia A Treatment, And Improvements To The Human Factor Viii Model, Ian Smith

Graduate Student Symposium

Blood coagulation factor VIII (FVIII), is a non-enzymatic cofactor which plays a crucial role in the formation of a stable blood clot. Absence or deficiency of FVIII results in the blood disorder hemophilia A; with symptoms including internal hemorrhaging and the inability to stop bleeding from open wounds. Treatment of hemophilia A relies on infusions of blood, plasma, or protein concentrates to replace FVIII. Unfortunately, approximately 30% of patients receiving replacement FVIII generate pathologic anti-FVIII inhibitory antibodies, which both reduce the effectiveness of the FVIII therapeutic and increase the severity of hemophilia A symptoms.

We have determined the molecular structure …

A Chemoenzymatic Approach For Synthesizing Polymeric Hemoglobin, Johann P. Sigurjonsson

Graduate Student Symposium

Polymerized hemoglobin (Hb) molecules have been shown to decrease previously observed adverse events associated with the administration of cell-free hemoglobin. To create these polymers, a method will be developed which employs the site specific ligation reaction of the sortase A enzyme from S. aureus. An Hb mutant (“αcpβ“) previously developed in our lab has been further modified by adding either the sortase recognition sequence, LPXTG, to the C-terminus of the α-subunit (s-αcpβ), or a tetraglycine motif, GGGG, to the N-terminus (n-αcpβ). Three types of sortase mediated ligation (SML) will be employed in this study. First, we will attempt to ligate …

Substrate Analogs For Characterizing The Substrate Tolerance Of S. Pneumoniae Srta, Orion Banks

Graduate Student Symposium

Bacterial sortases have been widely studied for their usefulness in protein modification, however, the variable substrate specificity and activity between homologs of these enzymes is not yet fully characterized. To attempt to further understand sorting signal recognition, we are working towards a substrate bound structure of sortase A from Streptococcus pneumoniae (SrtA_pneu). This enzyme displays a wide tolerance for alternate amino acids within the canonical LPXTG sorting motif. Our strategy involves a non-cleavable substrate analog that can be docked into the active site, allowing for elucidation of a structure displaying the key contacts that allow the enzyme …

Lipid Binding Studies Of Blood Coagulation Factor Viii C1 And C2 Domains, Rachel L. Blazevic

WWU Honors College Senior Projects

Blood coagulation factor VIII (fVIII) is an essential cofactor in the mammalian blood-clotting cascade. fVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for fIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, …

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 …

Smart Stimulation: Zoo Conservation For 21st Century Zoos, Lauren Retallack

WWU Honors College Senior Projects

According to a 1992 survey, "an estimated 102 million people, more than attend professional football, baseball, and basketball games combined, visit 162 accredited North American zoos and aquariums each year" (2). People frequent zoos for a variety of reasons, from entertaining children for a few hours, to learning about the wildlife which inhabits their region and foreign places. Regardless of intent, once at the zoo, visitors are presented with a unique opportunity to learn about conservation and the plights of endangered species. It is the job of zoo directors, keepers, staff, and volunteers to get people thinking about conservation while …

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 …