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The intracellular environment in which most proteins fold and function contains a range of biomolecules that results in significant volume exclusion, thus contrasting to the dilute buffer conditions common to most in vitro studies. In addition to intracellular macromolecular crowding, cells are ionic in nature, and although the Hofmeister series of ions has its origin in a work from 1888, much is still unclear concerning how small, charged ions affect protein properties. This thesis summarizes in vitro work assessing the effects of macromolecular crowding and small ions on the biophysical properties of a model protein -- Desulfovibrio desulfuricans flavodoxin. Flavodoxin is a small (15.7 kDa), single domain, cytoplasmic protein with alpha-helical and parallel beta-sheet secondary structural elements arranged in one of the five most common protein folds (the flavodoxin-like fold). Using a range of biophysical/spectroscopic methods (e.g., circular dichroism (CD), fluorescence, calorimetry, stopped-flow mixing) along with synthetic crowding agents (e.g., Ficoll and dextran), I have found that macromolecular crowding increases the secondary structural content of folded flavodoxin (toward that found in the crystal structure), increases flavodoxin thermal stability, and affects both the accumulation of a misfolded intermediate and the rate of proper protein folding. Collaborative in silico simulations employing Go-like modeling of apoflavodoxin in the presence of large, inert crowding agents agrees with my in vitro work and provides structural and mechanistic information with residue-specific resolution. We also found that small cations and anions in physiologically relevant concentrations (≤ 250 mM) increase flavodoxin thermal stability significantly. Both cations and anions in higher concentrations (300 mM-.75 M) affect oppositely charged proteins similarly suggesting that surface electrostatic charge plays only a minor role in mediating ionic effects on protein thermal stability. At all ion concentrations, ionic effects on protein stability are correlated to ion hydration (and thus the Hofmeister series). Our work suggests a dominant role for the peptide bond in coordinating ions at higher concentrations. This thesis work suggests that the crowded and ionic nature of the intracellular milieu can elicit changes to the structure, dynamics, stability, and folding mechanism of proteins which may not be captured in vitro using dilute buffer conditions.

The suddenly global outbreak of 2009 H1N1 Flu reminds human being the danger and severity of influenza virus. The gene of the new strain come from five different influenza viruses: North American swine influenza, North American avian influenza, human influenza, and two swine influenza viruses typically found in Eurasia. The recombination of gene in virus evolvement makes it more and more important to understand the evolutionary characteristics of influenza. Hemagglutinin (HA), embedded on the surface of influenza virus, is one of two virally-coded integral envelope proteins of the virus. The three primary functions of hemagglutinin (HA) include receptor binding, membrane fusion, and antigenic variation. Study of HA structure and its evolutionary mechanism is crucial to fully understand influenza virus. In the first chapter, a study of diversifying selective pressure on influenze B virus hemagglutinin was reported. All the positively selected sites were located in the four epitopes (120-loop, 150-loop, 160-loop and 190-helix) of HA, and all of them have been identified in previous studies. This supports a predominant role of antibody selection in HA evolution. In the second chapter, we studied positive selection analysis of 2009 HINT Flu. Among a subgroup of human A(H1N1) HAs between 1918∼2008, we found strong diversifying (positive) selection at HAI 156 and 190. We also analyzed the evolutionary trends at HAI 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA. Additional analysis of directional selection was also employed for H1N1 gene data.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3363. Adviser: Klaus Schulten. Includes bibliographical references (leaves 179-201) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3367. Adviser: David A. Agard.

Thesis (Ph.D.)--Indiana University, Dept. of Physics, 2008. / Title from PDF t.p. (viewed on Feb. 5, 2010). Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2130. Adviser: Sima Setayeshgar.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph.D.)--Lehigh University, 2009. / Adviser: James D. Gunton.

Our main research project is to develop an experimental system to understand the ribosomes as molecular motors. For this purpose, we designed and developed the single molecule ribosome motility assay. In this thesis, we discuss the related steps which we have taken to develop this assay: the instrumentation and four experiments. In the introduction, we illustrate the current experimental scheme and show the preliminary data of the single molecule ribosome translation. In the first two chapters, we discuss two interesting experiments dealing with stochastic problems. In the first chapter, we introduce the idea that the noise can be reduced in certain conditions. We show that the noise can be really reduced but only if added fluctuations have to drive the input into states with reduced intrinsic noise. In the second chapter, we investigate two-state processes experimentally using a simple physical system in which a microscopic bead is trapped in a double-well potential. We discuss how we can control the characteristics of the escape process by adding a periodic force and illustrate the characteristics in terms of the residence time distribution and the escape phase. As last two chapters, we discuss the elastic and structural properties of homopolymeric RNA, polyadenylic acids (poly(A)), polycytidylic acids (poly(C)), and polyuridylic acids (poly(U)). First, we investigate the elastic property of poly(U) as a random coil. We find that the force-extension data is well predicted by a classic worm-like chain model at high Na⁺ concentrations, whereas at low such concentrations the introduction of a scale-dependent persistence length is required. As single-stranded helices, poly(A) and poly(C) are studied. We stretch the molecules to induce the conformational transition from folded states to unfolded states and use the elastically coupled two-state model to acquire their structural information (about 9 bases per turn and 8 bases per turn, for poly(A) and poly(C) respectively, at neutral pH and 500 mM [Na⁺]) and the free energy differences between two states (20 pN·nm for poly(A) and 17.5 pN·nm for poly(C)).

We examine complex fluid systems where both translational and conformational degrees of freedom are present and focus on systems in which the interplay between the two sets of degrees of freedom is manifested in the macroscopic phase behaviour. We develop an efficient random lattice algorithm describing the translational degrees of freedom and analyze a series of microscopic models defined on a two dimensional fluid surface. Different degrees of complexity in the description of the microscopic coupling between the translational and conformational degrees of freedom allow us to study a variety of models related to pure lipid membrane and lipid-sterol membrane systems. / The phase equilibrium described by the models is calculated by use of Monte Carlo simulation techniques. The different models are shown to exhibit a rich phase behaviour. Depending on the specific model parameters, the phase transition associated with the conformational degrees of freedom is found to be either coupled to, or uncoupled from, that associated with the conformational degrees of freedom. / Specifically, the order-disorder transition of an Ising model defined on a fluid surface is shown to be of first order, when the two sets of degrees of freedom are strongly coupled. In contrast, the transition falls in the universality class of the two-dimensional Ising model when the two sets of degrees of freedom are weakly coupled. / We next analyze a model for pure lipid bilayers which is shown to exhibit a phase behaviour with different types of macroscopic coupling between the two sets of degrees of freedom. Depending on the strength of the microscopic interactions the lipid chain melting transition and the lattice melting transition may be either macroscopically coupled or uncoupled. / A related model for lipid-sterol mixtures is shown to provide a consistent interpretation of the various phases of lipid-cholesterol and lipid-lanosterol binary mixtures based on the microscopic dual action of the sterol molecule on the lipid-chain degrees of freedom. We discuss the results for the systems in the context of membrane evolution and suggest that evolution has tended to optimize the lipid-sterol interaction so as to stabilize optimally the mechanical properties of the membrane. Furthermore, a specific small-scale structure is identified and characterized in the liquid-ordered phase in lipid-cholesterol mixtures. This structure is found to be absent in lipid-lanosterol mixtures. / Finally, a model for membrane lysis gives evidence for the high mechanical stabilizing effect of cholesterol on the membrane. The inclusion of cholesterol is shown to inhibit lysis whereas lanosterol only has little stabilizing effect.