STRUCTURAL STUDIES OF TRANSCRIPTIONAL REPRESSOR DOMAIN (TRD) OF MBD1 AND MIDA PROTEINS

Ph.DDOCTOR OF PHILOSOPH

Models of the stability of proteins

Although the native conformation of a protein is thermodynamically its most stable form, this stability is only marginal. As a consequence, globular proteins have a certain amount of flexibility in their backbones which allows for conformational changes in the course of their biological function. In the course of this thesis, we study protein models at the edge of stability in different contexts: (1) First, we use molecular dynamics to determine the force needed to rupture a chain molecule (an unfolded protein) being stretched at constant loading rate and temperature. When all energy bonds of the molecule are identical, we find that the force F depends on the pulling rate r and temperature T according to F ~ const -- T 1/3|ln(r/T)|1/3 When a single weak bond is introduced, this result is modified to F ~ const -- T2/3|ln(r/ T)|2/3 This scaling, which is model independent, can be used with force-spectroscopy experiment to quantitatively extract relevant microscopic parameters of biomolecules. (2) Second, we study the structural stability of models of proteins for which the selected folds are unusually stable to mutation, that is, designable. A two-dimensional hydrophobic-polar lattice model is used to determine designable folds and these folds were investigated under shear through Langevin dynamics. We find that the phase diagram of these proteins depends on their designability. In particular, highly designable folds are found to be weaker, i.e. easier to unfold, than low designable ones. This is argued to be related to protein flexibility. (3) Third, we study the mechanism of cold denaturation through constant-pressure simulations for a model of hydrophobic molecules in an explicit solvent. We find that the temperature dependence of the hydrophobic effect is the driving force for cold denaturation. The physical mechanism underlying this phenomenon is identified as the destabilization of hydrophobic contact in favor of solvent separated configurations, the same mechanism seen in pressure induced denaturation. A phenomenological explanation proposed for the mechanism is suggested as being responsible for cold denaturation in real proteins

Undergraduate Research Opportunities Program 2011

Undergraduate Research Opportunities Program On August 9, 2011 the UNLV College of Sciences will celebrate the accomplishments of undergraduate students participating in the Summer 2011 Undergraduate Research Opportunities Program (UROP) and the Research Experience For Undergraduates (REU) Program. The public is invited to attend, beginning at 10:00 a.m. Please join us to view student research posters. Student research topics include: biomedicine and human health, Nevada\u27s fragile environment and ecosystems, climate change, stem cell research, microbiology, astrophysics, and many others. Over 25 UNLV undergraduates and a cohort of 25 undergraduates selected from colleges and universities across the nation will mark the completion of ten-week intensive research projects with UNLV faculty members, including projects in the life sciences, chemistry, physics, and other disciplines. This important program is funded by grants from the National Science Foundation (NSF), the National Institutes of Health (NIH), National Aeronautics and Space Administration (NASA), High Pressure Science and Engineering Center (HiPSEC) and the Nevada Idea Network of Biomedical Research Excellence Program (INBRE). The Undergraduate Research Opportunities Program (UROP) cultivates and supports research partnerships and invites undergraduates to work as the junior colleagues of faculty. The program offers the opportunity to work on cutting edge research—whether you join established research projects or pursue your own ideas. As participants, undergraduates engage in each phase of standard research activity: developing research plans, writing proposals, conducting research, analyzing data and presenting research results in oral and written form. The projects take place over the summer, and research can be done in any academic department or interdisciplinary laboratory. Projects can last for an entire semester, and many continue for a year or more. Students use their experiences to become familiar with the faculty, learn about potential majors, and investigate areas of interest. Participants gain practical skills and knowledge they eventually apply to careers after graduation or as graduate students. Most importantly, they become involved in state-of-the-art research. Students will present research posters, summarizing their work at UNLV. The students and their faculty mentors will be available to discuss and elaborate upon their scientific projects

In silico ligand fitting/docking, computational analysis and biochemical/biophysical validation for protein-RNA recognition and for rational drug design in diseases

Kaposi’s sarcoma-associated herpesvirus, is a double-stranded DNA γ - herpesvirus and the main causative agent of Kaposi’s sarcoma (KS). γ - herpesviruses undergo both lytic and latent replication cycles; and encode proteins that modulate host transcription at the RNA level, by inducing decay of certain mRNAs. Here we describe a mechanism that allows the viral endo-/exonuclease SOX to recognise mRNA targets on the basis of an RNA motif and fold. To induce rapid RNA degradation by subverting the main host mRNA degradation pathway SOX was shown to directly bind Xrn1. This may shed light as to how some viruses evade the host antiviral response and how mRNA degradation processes in the eukaryotic cell are involves in this

49th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 49th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Breckenridge, Colorado, July 22-26, 2007

NMR studies of the BamA complex proteins at high resolution

The β-barrel assembly machinery (BAM) complex is essential for the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria, with the membrane protein BamA acting as a catalyst for folding of OMPs in the outer membrane. Recently, structures of the BAM complex have been solved, displaying the molecular organization of the five proteins of the complex (BamABCDE). However, the mechanism by which BamA completes its insertase role is unclear. This PhD thesis focuses on the optimization of sample preparation and backbone assignment of the BamA β-barrel domain for solution NMR spectroscopy. Initial NMR spectra of the BamA β-barrel showed broad peaks with a low signal-to-noise ratio. This was likely due to a dynamic nature of the gate-region, as revealed by cysteine-scanning experiments. Therefore, as a first step to obtain a sample of BamA β-barrel suitable for NMR spectroscopy, buffer conditions were optimized. Then, in order to reduce the dynamics of the BamA β-barrel, a construct was designed with a C-terminal extension by nine residues. In addition to this extension, the G433A mutation in the gate-region was identified as to improve the quality of the NMR spectra. At that point, a combination of specific isotopic labeling and unlabeling in auxotrophic strains, triple-resonance experiments and 3D NOESY experiments allowed to obtain sequence-specific NMR resonance assignments of a large portion of the BamA β-barrel in LDAO micelles. The assignments revealed that some residues of the BamA β-barrel were found in different conformations that can be stabilized by the formation of a disulfide bond or by the C-terminal extension. Moreover, the crystal structure of the extended BamA β-barrel was determined, revealing a longer, and therefore more stable β-strand formed between the first and last strand of the barrel, explaining the stabilizing effect observed in its NMR spectrum. Additional work was performed on the soluble proteins of the BAM complex (BamBCDE). The expression and purification of BamB, BamC BamD and BamE was optimized and NMR spectra were recorded. BamD was found to be unstable once purified, and quickly precipitated, preventing to reach a molar concentration suitable for NMR spectroscopy. As a way to circumvent this issue, a hybrid construct of BamCD was prepared. The protein was able to reach high concentrations while keeping its stability. The fingerprint spectrum of BamCD was recorded and the peaks belonging to BamD overlapped with the peaks measured from a sample of individual BamD. This stabilized sample opens the possibility to obtain the sequence-specific assignments of BamD. Overall, this work resulted in the assignment of a large portion of the BamA β-barrel. As BamA is a potential target for new antibiotics, this assignment opens a way to perform NMR studies on BamA with substrates and ligands and understand the mechanical implications of their binding. Additionally, the dynamic nature of the BamA β-barrel was demonstrated by observing multiple conformations with solution-state NMR spectroscopy. In combination with the available assignment, it will be possible to observe the effects of binding molecules, mutations, or of the molecular environment on the conformational ensemble of BamA

Toward a molecular understanding of yeast silent chromatin : roles for H4K16 acetylation and the Sir3 C-terminus

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression. In budding yeast, silent chromatin is characterized by the loading of the Silent Information Regulatory (Sir) proteins (Sir2, Sir3 and Sir4) onto unmodified nucleosomes. This requires the deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions between Sir proteins forming the Sir2-3-4 complex. During my PhD thesis I sought to advance our understanding of these phenomena from a molecular perspective. Previous studies of Sir-chromatin interactions made use of histone peptides and recombinant Sir protein fragments. This gave us an idea of possible interactions, but could not elucidate the role of histone modifications in the assembly of silent chromatin. This required that we examine nucleosomal arrays exposed to full length Sir proteins or the holo Sir complex. In Chapter 2, I made use of an in vitro reconstitution system, that allows the loading of Sir proteins (Sir3, Sir2-4 or Sir2-3-4) onto arrays of regularly spaced nucleosomes, to examine the impact of specific histone modifications (methylation of H3K79, acetylation of H3K56 and H4K16) on Sir protein binding and linker DNA accessibility. The “active” H4K16ac mark is thought to limit the loading of the Sir proteins to silent domain thus favoring the formation of silent regions indirectly by increasing Sir concentration locally. Strikingly, I found that the Sir2-4 subcomplex, unlike Sir3, has a slight higher affinity for H4K16ac-containing chromatin in vitro, consistent with H4K16ac being a substrate for Sir2. In addition the NAD-dependent deacetylation of H4K16ac promotes the binding of the holo Sir complex to chromatin beyond generating hypoacetylated histone tails. We conclude that the Sir2-dependent turnover of the “active” H4K16ac mark directly helps to seed repression. The tight association of the holo Sir complex within silent domains relies on the ability of Sir3 to bind unmodified nucleosomes. In addition, Sir3 dimerization is thought to reinforce and propagate silent domains. However, no Sir3 mutants that fail to dimerize were characterized to date. It was unclear which domain of Sir3 mediates dimerization in vivo. In Chapter 3, we present the X-ray crystal structure of the Sir3 extreme C-terminus (aa 840-978), which folds into a variant winged helix-turn-helix (Sir3 wH) and forms a stable homodimer through a large hydrophobic interface. Loss of wH homodimerization impairs holo Sir3 dimerization in vitro showing that the Sir3 wH module is key to Sir3-Sir3 interaction. Homodimerization mediated by the wH domain can be fully recapitulated by an unrelated bacterial homodimerization domain and is essential for stable association of the Sir2-3-4 complex with chromatin and the formation of silent chromatin in vivo