Abstracts D1-D3

Category D. Protein Structure & Function

 

D1. Protein Production Group: From Primers to Proteins, and Beyond

*Philip Gao, *Anne Cooper, Na Zhang, Anna Chang, Linsee Addington
Protein Structure Group, Center of Biomedical Research Excellence in Protein Structure and Function, 2034 Becker Drive, The University of Kansas, Lawrence, KS, 66047

The Protein Production Group is one of the three core laboratories of the Kansas COBRE in Protein Structure and Function and it provides services at all stages of the vector and mammalian stable cell line construction, protein expression, purification and modification processes, for functional (catalytic or biological), binding (small ligand or macromolecules using Biacore), structural (X-ray, NMR), and high throughput (HTP) screening studies. Both prokaryotic and eukaryotic proteins can be produced in E. coli, yeast, insect cell, mammalian and cell-free expression systems. The facility also has the capacity for expanding on existing purifications schemes for large scale preparations. Most of the preparations and purifications are conducted in conjunction with automated equipment to ensure precision and efficiency.  The Protein Production Group’s objective is to over-express and purify properly folded functional proteins in quantities sufficient for functional studies (catalytic or biological), binding assays (small ligand or macromolecular), structural analysis (X-ray, NMR), and high throughput (HTP) screening. In addition, the facility also provides advice on the generation and analysis of proteins depending on their physicochemical properties and the needs of the investigator.

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D2. Structural Studies of HDAC 4: MEF 2 for Lead Identification of Protein-Protein Interaction Inhibitors

Nurjahan Mehzabeen, Na Zhang, Anne Cooper, Phillip Gao and Scott Lovell
Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas.

Recent studies have shown that transcriptional dysregulation is a major process involved in the pathology of Huntington’s disease, an inherited neurodegenerative disorder characterized by the presence of a mutant form of the huntingtin protein. It has been suggested that the mutant huntingtin protein affects histone acetyltransferase activity resulting in a reduction in histone acetylation causing repressed transcription of particular genes. One approach to combat this transcriptional dysregulation is to inhibit the catalytic activity of histone deacetylases, such as HDAC4, thereby allowing unregulated expression of certain genes in Huntington’s disease patients. However, the effectiveness of current inhibitors of HDAC4 is limited due to their toxicity. An alternative approach would be to inhibit the interaction between HDAC4 and respective protein partners (ex., MEF2) that negatively regulate transcription. A structural model of the HDAC4:MEF2 complex is anticipated to be valuable to a structure-based design of small molecule modulators of these protein:protein interactions. In an effort to identify lead candidate compounds that target the HDAC4:MEF2 complex, the Protein Structure and Protein Production Laboratories, at the University of Kansas, are working to characterize the molecular interactions between MEF2 and HDAC4.  To accomplish this goal, we have expressed and purified constructs of HDAC4 and MEF2 for analysis by surface plasmon resonance and X-ray crystallography.  The initial results of this project are highlighted. 

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D3. X-ray Structure Of Human Cytochrome P450 1A1 And Insights Into Function From Docking Studies

Agnes A. Walsh1, Grazyna D. Szklarz2, and Emily E. Scott1
1The Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS
2Deparment of Basic Pharmaceutical Sciences, West Virginia University P.O. Box 9530, Morgantown, WV

Cytochrome P450 1A1 (CYP1A1) is an extrahepatic monooxygenase involved in the metabolism of several drugs and endogenous substrates, as well as the bioactivation of environmental contaminants.  This enzyme is particularly well known for its ability to bioactivate polycyclic aromatic hydrocarbons into carcinogens, such as benzo[a]pyrene in tobacco smoke.  However, no experimental structures have been available to facilitate an understanding of CYP1A1 enzymatic activity.  Thus, a truncated, His-tagged form of the human cytochrome P450 1A1 enzyme was expressed recombinantly and used to determine an X-ray crystal structure in complex with the inhibitor a-naphthoflavone (ANF) at 2.6 Å.  The overall CYP1A1 structure corresponds to other mammalian cytochrome P450 enzymes, but with a disruption of the F helix presently only observed in CYP1 family enzymes.  The planar polycyclic ANF binds within a narrow, enclosed active site sandwiched between the I helix and a key Phe224 residue that participates in p-p stacking with the benzochromenone core.  The ANF 2-phenyl substituent is oriented toward the catalytic heme iron.  Docking typical CYP1A1 ligands, including resorufins, phenacetin, resveratrol, and benzo[a]pyrene, into the X-ray structure reveals binding within the same plane as ANF and indicates steric features that may be responsible for ligand orientation within the active site.  Structures of the other two human CYP family 1 enzymes, the hepatic CYP1A2 and extrahepatic CYP1B1 enzymes, have also been determined with ANF, allowing for direct comparisons. Structural similarities and differences among these three enzymes and the results from ligand docking suggest active site topologies that may underlie their distinct functional capabilities.

Funding provided by the National Institutes of Health (GM076343).

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