No description available.

There are two contributions of this thesis. The first contribution, described in chapters one through six, involves studing the relationship between the protein packing structure of bacteriorhodopsin (bR) and its function as a proton pump. In 2002, a novel crystallization method published by Bowie and Farham resulted in an unusual antiparallel monomeric packing structure of bicelle bacteriorhodopsin (bcbR) crystals, the spectroscopic properties of which had not been studied. In this thesis, these bicelle bR crystals are investigated to better understand how the changes in the protein tertiary structure affect the function. Specifically: Does the retinal Schiff base retain its ability to isomerize in this unusual protein packing structure of bR? How is the hydration of its binding pocket affected? Does the protein retain the ability to undergo the photocycle and pump protons? If so, how are the rates of the deprotonation/reprotonation of the Schiff base affected by the antiparallel monomer packing structure of the protein? Is Asp85 still the proton acceptor during the deprotonation process of the photocycle? The second contribution of the thesis, described in chapter seven, describes the surface attachment and growth of the biofilm formed by the pathogenic bacterium Streptococcus pneumoniae using attenuated total reflection/Fourier transform infrared spectroscopy (ATR/FTIR). This organism was chosen for its clinical significance; it is one of the organisms suspected in forming biofilms in individuals who develop otitis media, one of the most common causes of ear infections of childhood. In contrast to previous ATR/FTIR experiments examining the formation of biofilms on surfaces, this method is unique in that it combines two techniques - ATR/FTIR and Epifluorescence microscopy which when used together allow for the simultaneous monitoring of the IR spectrum of the S. pneumoniae biofilm as it develops and as provides a method for quantifying total and viable cell counts at various stages during the development.

This work aimed at studying biophysical properties of two membrane proteins, one of potential nanotechnological use, bacteriorhodopsin, and one potential drug target, the NTS1 neurotensin receptor, at the single molecule scale. Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) of the halophilic organism Halobacterium salinarium. It is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of its retinal chromophore. Its stability, as well as its photoactivity remaining in dry protein layers, has made BR an attractive material for biomolecular devices. Numerous studies have been published on this topic; however, they have all used BR within the PM, on relatively large (µm-wide) surfaces. Here, conducting-probe atomic force microscopy (C-AFM) analysis was performed after removing most of the membrane lipids. For the first time, it was shown that the molecular conductance of BR can be reversibly photoswitched with predictable wavelength sensitivity. Intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine which, combined with partial delipidation, generated protein trimers homogenously orientated on the surface. Numerous controls using biophysical (SPR, ellipsometry, Kelvin-probe AFM) and chemical (photocurrent, cyclic voltammetry) techniques confirmed the wavelength specificity of the photoswitch, the anchoring role of the mutation and the homogenous orientation of the protein on the gold surface. Neurotensin is a brain and gastrointestinal 13 amino acid peptide acting as a neuromodulator in the central nervous system and as a hormone in the periphery. Its wide range of biological activities is primarily mediated through its binding to the neurotensin type 1 receptor (NTS1). NTS1 expressed in E.coli was purified and inserted into 100 nm brain polar lipid liposomes in a conformation which retained its ligand-binding capabilities. Initial AFM characterisation was performed as a prelude for ligand-receptor interaction studies, including high resolution imaging, force spectroscopy and solid state NMR approaches.

Polihidroxialcanoatos (PHAs) são polímeros produzidos por diversas bactérias como material de reserva de carbono e energia, apresentando propriedades termoplásticas comparáveis às dos plásticos de origem petroquímica, além da vantagem de ser totalmente biodegradáveis. A bactéria Cupriavidus necator DSMZ 545 é considerada o organismo modelo para a produção de PHA, possuindo a capacidade de utilizar CO2 como fonte de carbono, porém existem sérias limitações para o seu cultivo autotrófico. Visando otimizar a capacidade autotrófica desta bactéria, foi realizada neste trabalho a clonagem e expressão do gene da bacteriorodopsina (bop) de Halobacterium salinarum em C. necator. Esta proteína de membrana é capaz de converter a energia luminosa em um gradiente de prótons, que pode ser utilizado pela célula para produzir ATP. Obtiveram-se clones recombinantes de C. necator, que, sob iluminação, apresentaram maior produção de ATP, assim como produção aumentada de PHA, sem alterações no crescimento celular. / Polihydroxyalcanoates (PHAs) are polymers produced by several bacteria as carbon and energy storage materials, which show comparable thermoplastic properties with plastics from petrochemical origin and present the advantage of being completely biodegradable. The bacterium Cupriavidus necator DSM 545 is considered as a model organism for PHA production, being able to produce PHA utilizing CO2 as the carbon source, although there are severe limitations for its autotrophic cultivation. In the present work, with the purpose of optimizing the autotrophic capability of this bacterium, the cloning and expression of the Halobacterium salinarum bacteriorhodopsin gene (bop) was undertaken in C. necator. This membrane protein is able to convert light energy in a proton gradient that can be utilized by the cell for ATP production. Upon illumination, the C. necator recombinant clones showed an increase in ATP production, as well as an increase in PHA production, and no alterations in cell growth.

E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.

No description available.

The first section of this thesis concerns the study of interactions between the intense local plasmonic field generated by nanostructure and a well known photosynthetic protein system, bacteriorhodopsin (bR). bR is a membrane protein responsible for proton transport. Among the many intermediates formed upon photoexcitations, two of the most relevant have been studied. The intermediates under studies were I460 and M412, and their decay dynamics were measured in presence of the plasmonic field generated by the excitation of their surface electrons using visible photons. Both intermediates decay lifetime were affected when the plasmonic field was turned on, and it was verify that thermal effect were not the source of the change in dynamic. The second part concerns the investigation of third-order nonlinearity of a series of extended conjugated squaraine dyes in the telecommunication spectral region. Their nonlinearity is measured via Degenerate Four Wave Mixing and Z-scan as function of the dyes increasing conjugation length and number of squarylium groups. The dyes produced large real and imaginary values for the third order nonlinearity in the 1300-1500 nm range which makes them attractive for optical limiting type of applications.

No description available.

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.