Friday May 4th – Presentations

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Spin-Labeling Magnetic Resonance Applications in the Glycine Riboswitch

Gail E. Fanucci, Michelle Ehrenberger, Jacqueline M. Esquiaqui.

Department of Chemistry, University of Florida

10:15 AM

Our lab utilizes site-directed spin labeling magnetic resonance (SLMR) to characterize motion and flexibility associated with function and malfunction. Here we will discuss our latest activates related to strategies to characterize conformational changes in the glycine riboswitch with particular focus on the leader-linker region compared to the glycine binding region of the second aptamer. We are able to show that different regions of this large dynamic RNA are pre-formed in solution and stabilized by either monovalent or divalent ions. To date, it appears that the riboswitch regions preform prior to binding glycine, possibly indicating that a truncated riboswitch that does not contain the expression platform does not completely recapitulate the true conformational landscape of the in vivo messenger RNA species.

Computational Study of alkene reduction using Old Yellow Enzyme

Sunidhi Lenka, M. Pilar Buteler, Robert W. Powell, III, Jon D. Stewart, Adrian E. Roitberg

University of Florida

10:50 AM

Old Yellow Enzymes (OYE) are a class of enzymes known for reduction of electron deficient alkenes with high stereoselectivity, leading to generation of synthetic intermediates for flurbiprofen (non-steroidal anti-inflammatory drugs) and pregabalin etc. The potential of OYE to generate chiral centers has led to various protein engineering approaches to obtain high yield of stereoselective products.

The present research focuses on OYE 1 and OYE 3, which are different homologs of the old yellow enzymes having high sequence identity. Previous experimental studies of OYE 1 indicated a change in stereoselectivity of product, by mutation of Trp 116 for substrate (S)- carvone and (R)- carvone. Further studies showed that OYE 3 produced different enantiomeric excess products for some substrates compared to OYE 1. Comparing the active site of both proteins, the only difference was found at position 296, with Phe for OYE 1 and Ser for OYE 3. This residue is present in loop 6 (289-309) which plays a significant role in reactant accommodation. Hence, studying the dynamic properties of this loop and its effect on stereoselectivities would provide a deeper perspective of the enzyme properties.

Molecular dynamics simulations using the AMBER GPU suite has been used to rationalize the difference in stereoselectivities and develop specific protein engineering methodology to generate products. The system has been studied along a specific reaction coordinate using umbrella sampling, in order to sample different binding orientations of the substrate. It has been found that, loop 6 is much more flexible and solvent exposed for OYE 3 than OYE 1. Hence, it provides more freedom for the substrate to orient in the active site. The results from our group are consistent with the experimental data leading us to further investigate the catalytic properties of the active site.

Structural characterization of the Rous Sarcoma Virus capsid protein in its tubular assembly and simulations of the self-assemblies of the HIV capsid protein

Jaekyun Jeon, Ivan Hung, Alok K. Mitra, Ambroise Desfosses, Xin Qiao, Daniel Huang, Peter L. Gor’kov, Rebecca C. Craven, Richard L. Kingston, Zhehong Gan, Fangqiang Zhu, and Bo Chen

Department of Physics, University of Central Florida

11:10 AM

The capsid proteins (CAs) of ortho-retroviruses share a common tertiary fold but form distinct capsids. They are promising antiviral drug targets and templates for versatile nano-assemblies. However, they are tough to characterize due to the strong polymorphism. In this work, solid state NMR was applied to characterize the CA tubular assembly of Rous sarcoma (RSV), a prototype of retrovirus. A novel resonance assignment strategy was developed that exploits the well-resolved NCACX spectra to facilitate the assignment of congested NCOCX spectra, which led to a nearly complete assignment (234 residue out of the 237-residue protein). Based on this, site-specific dynamics and secondary structural information were determined. Combining with constraints from cryo-EM, we established an atomic resolution model of the tubular assembly by molecular dynamics flexible fitting. Our model shows that significant structural rearrangements take place at flexible loops and the 310 helix regions, while the rest of the protein retains its structure upon assembly. The analyses of our model suggests the assembly polymorphism is attributed to the disorder of the trimer interface between C-terminal domains. In addition, the different contact angles between helices at assembly interfaces of tubular and planar assemblies for HIV and RSV CA. Based on simulations of our novel coarse grain model, it suggests the two systems undergo different assembly pathways.