Saturday May 6th – Presentations

In by admin


Joseph M. Pennington and M. Elizabeth Stroupe

Department of Biological Science and Institute of Molecular Biophysics
Florida State University

01:30 PM
to 02:05 PM

Siroheme is the modified tetrapyrrole used by siroheme-dependent sulfite and nitrite reductases (SiR/NiRs) in catalyzing the six-electron reduction of sulfite to sulfide or nitrite to ammonia. Proteobacteria like Escherichia coli or Salmonella typhimurium use a single gene product to synthesize siroheme, CysG. CysG is the product of expressing a gene fusion between a C-terminal methyltransferase (CysGA) and an N-terminal bifunctional dehydrogenase/ferrochelatase (CysGB). CysGB is a three-domain homodimer composed of an N-terminal Rossmann fold that binds NAD+, a four-stranded β-sheet dimerization domain, and a C-terminal α-helical domain. Together, these three domains form an X-shaped molecule, where the Rossmann fold from one subunit and the helical domain from the other subunit carve out a large cavity. Although this large cavity is suggestive of a porphyrin binding site, we do not yet know the mechanism for positioning precorrin-2 for dehydrogenation at C12. Additionally, despite extensive mutagenesis in an attempt to identify a metal ligand, it is unclear how iron is selected and then inserted in the final step. We have successfully determined the X-ray crystallographic structure of S. typhimurium CysG bound to sirohydroclorin and siroheme in the dehydrogenase/chelatase domain that suggest a mechanism for hydride abstraction and iron chelation.


Isabel Askenasy1, Rachel Andrews2, and M. Elizabeth Stroupe1,2

1 Department of Biological Science, Florida State University, Tallahassee FL 32306 - 4295, USA.
2 Institute of Molecular Biophysics, Florida State University, Tallahassee FL 32306 - 4380, USA.

02:05 PM
to 02:25 PM

Sulfite Reductase (SiR) is a key enzyme in sulfur assimilation, performing the six electrons reduction of sulfite to sulfide. In Enterobacteria, SiR is dodecameric complex with eight copies of the α subunit, or Flavoprotein, and four copies of the β subunit, or Hemoprotein. We aim to understand how electron transfer occurs in the context of SiR’s unique stoichiometry and structure. From our biochemical analysis, we have discovered that SiR’s subunits share two interfaces: one functional and one structural. Surprisingly, the structural interface is far from where electron transfer is known to occur. Therefore, we hypothesize that the functional interface occurs through a transient interaction between the subunits. To test our hypothesis, we synthesized four Flavoprotein point variants. The variations were introduced in the structural interface, the functional interface or the flexible region that connects the domains where the interfaces are located. We then measured binding affinities using ITC and enzyme activity through in vivo and in vitro experiments. Electron transfer is, indeed, independent from tight binding.


Kevin Jeanne Dit Fouque,1 Luis M. Salgueiro,2 Renzhi Cai,2 Wei Sha,2 Andrew V. Schally,2 and Francisco Fernandez-Lima.1

1 Department of Chemistry and Biochemistry, Florida International University, Miami, FL.
2 Departments of Pathology and Medicine, Divisions of Hematology/Oncology and Endocrinology, Miller School of Medicine, University of Miami, Miami, FL.

02:25 PM
to 02:45 PM

Growth hormone – releasing hormone (GHRH) is a 44 residue hypothalamic peptide hormone that specifically stimulates the secretion and release of growth hormone from the anterior pituitary gland upon binding to its receptor. GHRH and its receptor are expressed in many human cancer cell lines and tumors as well as in other tissues including myocardium and pancreatic β cells. Understanding the action of GHRH on its target cells is important for the use of synthetic GHRH analogs, with prolonged half-lives, which may provide a promising scaffold for drug development. In the present work, a set of three GHRH agonists, MR-356, MR-406 and MR-409 and three GHRH antagonists, MIA-602, MIA-606 and MIA-690 were investigated using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) to study the kinetically trapped intermediates species of these analogs as a function of the starting solution conditions (native conditions vs denaturing conditions) and as a function of the collision induced activation (CIA) prior the TIMS-MS measurements. Comparison between GHRH (1-29) and its synthetic agonist and antagonist conformational spaces and dynamics are described as a way to gain a better understanding of the conformation involved in the biological activity.


Alyssa Garabedian1; Prem Chapagain2; Fenfei Leng1,3; and Francisco Fernandez-Lima1,3*.

1 Department of Chemistry and Biochemistry, Florida International University, Miami, USA
2 Department of Physics, Florida International University, Miami, USA
3 Biomolecular Science Institute, Florida International University, Miami, USA

03:00 PM
to 03:20 PM

The mammalian high mobility group AT-hook 2 (HMGA2) is an architectural transcription factor traditionally characterized as being unstructured. This disordered-to-ordered transition has implicated HMGA2 as a protein actively involved in many biological processes. Additionally, the abnormal expression of HMGA2 has been linked to a variety of health problems including diabetes, obesity and oncogenesis. In this work, for the first time we take advantage of trapped ion mobility spectrometry coupled to mass spectrometry to study the conformational space of HMGA2 and their DNA-complexes, not accessible using traditional structural biology tools. Our experiments showed that HMGA2 monomer can exist as multiple kinetic intermediates with varying tridimensional structure (folded to unfolded transitions), regardless of the starting solution conditions. Moreover, HMGA2 complex with a short 22 base hairpin DNA can be preserved in the gas phase and shows multiple kinetically trapped conformational states. In contrast, HMGA2 complex with a longer 50 base hairpin DNA complex can also be preserved in the gas-phase and the number of kinetically trapped conformational states is greatly reduced by the higher number of intramolecular interactions (e.g. binding sites). When combined with molecular dynamics, for the first time, candidate structures were proposed for the HMGA2 and HMGA2-DNA kinetically trapped intermediates.

Conformation plasticity of the N-terminal intrinsically disordered region of ChiZ memrbane protein

Cristian A. Escobar1,2 and Timothy A. Cross1,2

1 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA
2 National High Magnetic Field Laboratory, Tallahassee, FL, USA

03:20 PM
to 03:40 PM

ChiZ is a transmembrane protein from Mycobacterium tuberculosis involved in cell division regulation. It was proposed that the N-terminal cytoplasmic domain is essential for cellular activity even though it is intrinsically disordered. Using solution NMR, it was possible to identify two regions in the N-terminal domain with different dynamics, amide proton exchange rates and tendencies to form compact structure. Interestingly, one of these regions is conserved, which suggests these differences are important for function. To assess the relevance of these differences, the N-terminal domain was studied in the presence of liposomes and in the full length protein using a combination of solution and solid state NMR. The N-terminal domain is able to bind liposomes, but it remains highly dynamic, even in the reconstituted full length protein. Paramagnetic relaxation enhancement experiments showed that the N-terminal domain interaction with lipids is mediated by electrostatic interactions between arginine side chains and lipid head groups. However, the conserved region has a lower tendency to interact with lipids. This data suggests that the loose binding of the N-terminal region to lipids may facilitate the sampling of conformations that may be required for function. Speculation as to this function in the cytoplasm will be presented.

The dock-and-coalesce mechanism for the association of a WASP disordered region with the Cdc42 GTPase

Li Ou, Megan Matthews, Xiaodong Pang, and Huan-Xiang Zhou

Department of Physics and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA

03:40 PM
to 04:15 PM

Intrinsically disordered proteins (IDPs) play key roles in signaling and regulation. Many IDPs undergo folding upon binding to their targets. We have proposed that coupled folding and binding of IDPs generally follow a dock-and-coalesce mechanism, whereby a segment of the IDP, through diffusion, docks to its cognate subsite and, subsequently, the remaining segments coalesce around their subsites. Here, by a combination of experiment and computation, we determined the precise form of dock-and-coalesce operating in the association between the intrinsically disordered GTPase binding domain (GBD) of the Wiskott-Aldrich Syndrome protein (WASP) and the Cdc42 GTPase. The association rate constants (ka) were measured by stopped-flow fluorescence under various solvent conditions. ka reached 107 M-1s-1 at physiological ionic strength and had a strong salt dependence, suggesting that an electrostatically enhanced, diffusion-controlled docking step may be rate-limiting. Our computation, based on the transient-complex theory, identified the N-terminal basic region of the GBD as the docking segment. However, several other changes in solvent conditions provided strong evidence that the coalescing step also contributed to determining the magnitude of ka. Addition of glucose and TFE and an increase in temperature all produced ka values much higher than expected from the effects on the docking rate alone. Conversely, addition of urea led to ka values much lower than expected if only the docking rate was affected. These results all pointed to ka being approximately two thirds of the docking rate constant under physiological solvent conditions. The dock-and-coalesce mechanism allows WASP and other IDPs to code electrostatic complementarity into the docking segment to gain binding speed and use additional interactions formed by the coalescing segments to reinforce binding affinity.