abstract: Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase. / Dissertation/Thesis / Ph.D. Chemistry 2011

Gene regulation by transcription factors are one of the key processes that are important to sustain all kinds of life. In the prokaryote Escherichia coli this has shown to especially crucial. The operator sequence to which these transcription factors bind to are very small in comparison to the whole genome of E. coli, thus the question becomes how these proteins can find these sequences quickly. One particularly well-studied transcription factor in this regard is the lac repressor. It has been shown that this transcription factors finds its operators faster than the limit of three dimensional diffusion. The leading model for how the repressor does that is facilitated diffusion and this model has gained more experimental evidence, particularly using single-molecule fluorescence microscopy. This study aimed at measuring the unspecific binding time between the lac repressor and DNA in vivo, but in the end the project evolved to trying to establish a single-molecule tracking assay of the repressor in vivo. In this study a mutant of the repressor was expressed and purified, labelled with a synthetic fluorophore, electroporated into E. coli and tracking was performed under a microscope. One of the three types of experiments were partially analysed with an image analysis software. Unfortunately, analysis was not completed for all experiments which made it difficult to compare the results. In the end the data was compared by eye while also using the results from image analysis. With slight optimism it can be concluded that the assay worked, but it needs more development.

The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) cause food poisoning in humans. Salmonellae also cause disease in animals of economic importance like poultry and cattle. Treatment of diseases caused by these notorious pathogens is becoming more and more difficult because of the emergence of drug resistant strains. Thus, it is vital to understand the virulence mechanisms of Salmonella which can lead us to potential drug targets and also help us design effective vaccines. Salmonella has evolved many strategies to enter the host, to evade intracellular and extracellular antimicrobial activities of the host and to extract nutrition in the stringent and hostile environment of the host. These strategies have enabled Salmonella to survive and multiply in the host making it a successful pathogen. Present study deals with four such survival strategies of Salmonella. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever caused by serovar Typhi in humans. This serves as a good model system to study and understand the pathogenesis of Salmonellae. This model system has been used throughout this study. In the present thesis attempts have been made to identify some novel survival strategies of Salmonella. The thesis is divided into five chapters. Chapter 1 gives an introduction into the basic biology of these notorious pathogens. The diseases caused by Salmonellae are introduced in this chapter. Typhoid fever is discussed in detail covering its epidemiology, clinical features, diagnosis, treatment and prevention. Next section covers the virulence determinants of Salmonella. In this section, Salmonella pathogenicity islands are discussed in detail. This chapter concludes with an overview of molecular pathogenesis of Salmonella covering its invasion strategy and its dangerous life inside the host cell. Salmonella stays and multiplies inside a specialized endosomal compartment of the host cell known as Salmonella-containing vacuole (SCV). It is believed that Salmonella multiplies inside SCV resulting in single big vacuole containing multiple bacteria. The results of Chapter 2 challenge this notion. Using transmission electron microscopy and confocal laser scanning microscopy we show that SCV also divides along with the division of Salmonella resulting in multiple SCVs containing single bacterium per vacuole. We also show that this division is mediated by the molecular motor dynein. This chapter concludes with a discussion on the advantages of SCV division with respect to Salmonella. Successful intracellular pathogens must have some strategy either to avoid lysosomal fusion or to endure the toxic molecules of lysosomes. In case of Salmonella, it is well accepted that SCV-lysosome fusion is blocked. However, the exact mechanism of this process is still unclear. The results of Chapter 3 enhance our understanding of this issue. This chapter explores an interesting possibility of Salmonella reducing the lysosomal number and thereby reducing the chances of SCV-lysosome fusion. Using flowcytometry and confocal laser scanning microscopy, we show that Salmonella decreases the number of acidic lysosomes in murine macrophages. Thus, our results suggest that there is an imbalance in the ratio of vacuoles to acidic lysosomes which decreases the probability of SCV-lysosome fusion thereby helping Salmonella avoid lysosomes. Multicellular organisms use various defense strategies to protect themselves from microbial infections; production of antimicrobial peptides (AMPs) is one of them. Being cationic in nature, AMPs interact and cause pores in the bacterial membrane eventually killing the bacteria. Pathogenic micro-organisms like Salmonella have evolved many strategies to counteract the AMPs they encounter upon their entry into the host systems. S Typhimurium genome has a gene cluster consisting of yejA, yejB, yejE and yejF genes which encode a putative ABC transporter. Chapter 4 deals with the detailed characterization of these genes. Our study shows that these genes constitute an operon. We have deleted the yejF gene which encodes the ATPase component of this putative ABC transporter. The ΔyejF strain showed increased sensitivity to AMPs like protamine, melittin, polymyxin B and human defensins and was compromised to proliferate inside activated macrophages and epithelial cells. In murine typhoid model, the ΔyejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs and contributes to the virulence of Salmonella. An important biochemical property of Salmonella that distinguishes it from the closely related E. coli is its inability to ferment lactose. In E. coli, lactose fermentation is carried out by the products of lac operon which is regulated by a repressor encoded by lacI. Salmonella does not have the lac operon and lacI. It has been proposed that S.enterica has lost lac region (lacI and lacZYA) during its evolution. Chapter 5 deals with the evolutionary and physiological significance behind the loss of lac region by S.enterica. We show that expression of LacI in S. enterica suppresses its virulence by interfering with the expression of SPI-2 virulence genes. We also observed that the genome of S. bongori which does not have the virulence genes of SPI-2 has a homologue of LacI. Our results suggest that presence of lacI has probably hindered the acquisition of virulence genes of SPI-2 in S. bongori, whereas absence of lacI has facilitated the same in S. enterica making it a successful systemic pathogen. Thus, lacI has played a remarkable role in the evolution of Salmonella virulence. Brief summary of four studies that are not directly related to survival strategies of Salmonella are included in Appendix. First two studies analyze molecular evolution of SPIs to understand the mechanism of host specificity in Salmonella and the last two studies explore the signaling of lipopolysaccharide (LPS) derived from Salmonella.

Evolución dirigida de proteínas: La evolución dirigida es una técnica que nos permite explorar funciones enzimáticas que no son requeridas en el ambiente natural. Esta técnica, simula procesos genéticos naturales y de selección. Esta estrategia se utiliza cuando un diseño racional es muy complicado. Consiste en una repetición de ciclos de diversificación y selección que llevan a la acumulación de mutaciones benéficas. Aquí se presenta dos ejemplos de evolución dirigida con los cuales se ha trabajado directamente: la ADN polimerasa del organismo  Thermus aquaticus usada comúnmente en PCR, y la proteína LacI que regula la expresión de genes usados para el metabolismo de lactosa en E. Coli. / Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.