In this study, a Monte Carlo simulation was designed to observe the self-assembly of the HIV-1 capsid protein. The simulation allowed a coarse grained model of the capsid protein with defined interaction sites to move freely in three dimensions using the Metropolis criterion. Observations were made as to which parameters affected the assembly the process. The ways in which the assembly were affected were also noted. It was found that proper dimerization of the capsid protein was necessary in order for the lattice to form properly. It was also found that a strong trimeric interface could be responsible for double-layered assemblies. Further studies may be conducted by further varying of parameters or reworking the dynamics of the simulation. The possible causes of curvature within the assembly still need to be researched further.

Integrins are large heterodimeric (αβ) cell surface receptors that play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are ‘activated’ to a high affinity state by the formation of an intracellular complex between the membrane, the integrin β-subunit tail and talin, a process known as ‘inside-out activation’. The head domain of talin, a FERM domain homologue, plays a vital role in the formation of this complex. Recent studies also suggest that kindlins act in synergy with talin to induce integrin activation. Despite much available structural and functional data, details of how talin activates integrins remain elusive. In this thesis a multiscale simulation approach (using a combination of coarse-grained and atomistic molecular dynamics simulations) together with NMR experiments were employed to study talin-mediated integrin inside-out activation. A number of novel insights emerged from these studies including: (i) the crucial role of negatively charged lipids in talin/membrane association; (ii) a novel V-shape conformation of the talin head domain which optimizes its interactions with negatively charged lipids; (iii) that interactions of talin with negatively charged moieties in the membrane orient talin to optimize interactions with the β cytoplasmic tail; (iv) that binding of talin to the β cytoplasmic tail promotes rearrangement of the integrin TM helices and weakens the integrin α/β association; and (v) that an increase in the tilt angle of the β integrin TM helix initiates a scissoring movement of the two integrin TM helices. These results, combined with experimental data, provide new insights into the mechanism of integrin inside-out activation. The same multiscale approach was used to demonstrate the crucial role of the Gx3G motif in the packing of the integrin transmembrane helices. Using recent structural data the integrin/talin complex was modelled and inserted in bilayers which resemble the biological plasma membrane. The results demonstrate the dynamic nature of the integrin receptor and suggest that the integrin/talin complex alters the lipid organization and motion in the outer and inner bilayer leaflets in an asymmetric way and that diffusion of lipids in the vicinity of the protein is slowed down. The work in this thesis demonstrates that multiscale simulations have considerable strengths when applied to investigations of structure/function relationships in membrane proteins.

A Thermotoga marítima (Tm) é uma bactéria que vive em temperaturas na faixa dos 65 até 90°C, com temperatura ótima do redor dos 80°C. A proteína TM1030 de Tm, é um regulador transcricional da família TetR (Tetracycline repressor protein) reguladores da expressão génica das proteínas TetA e TetB (Tetracycline resistance protein). Neste trabalho se rodarem 200ns de trajetória de dinâmica molecular a três temperaturas (293, 323 e 353K) da proteína TM1030 (PDB-1Z77) usando o pacote GROMACS com o potencial Amber99 e solvente explicito numa caixa cúbica com 90Å de comprimento, observando que RMSD da estrutura média da trajetória é menor em relação à estrutura cristalográfica, além disso que num primer momento esse RMSD tem uma mudança grande e que se estabiliza com uma maior velocidade nas maiores temperaturas. Também foi feito um analise de modos normais na mesma estrutura usando o mesmo potencial, mas com solvente implícito, usando o modelo GBSA, minimizando a estrutura até ter um coeficiente de força média de 6,4x10-8J·mol-1·cm-1 que assegura um bom mínimo local. Das trajetórias simuladas a partir das 6 menores frequências se achou uma relação com os movimentos observados nas dinâmicas moleculares e os esperados na transição alostérica entre as duas estruturas cristalográficas. Finalmente se calculam os fatores de temperatura das três trajetórias de dinâmica molecular, observando que seus esses fatores de temperatura aumentam com o aumento da temperatura, contrario do esperado da cristalografia onde diminuam com o aumento da temperatura do sistema. / The Thermotoga maritima (Tm) is a bacterium who can lives at temperatures of 65 to 90°C, with optimum temperature around of 80°C. The TM1030 protein of Tm is a transcriptional regulator from TetR family (Tetracycline repressor protein) regulators of gene expression of the TetA and TetB protein (Tetracycline resistance protein). In this work 200ns of molecular dynamics trajectory was run at three temperatures (293, 323 and 353K) of TM1030 protein (PDB-1Z77) using GROMACS package with Amber99 potential and explicit solvent in a cubic box with length 90A, noting that RMSD of the average structure of the trajectory is smaller with respect to the crystallographic structure, in addition, in a first time this RMSD have a large change and stabilizes at a higher speed at higher temperatures. There was also an analysis of normal modes on the same structure using the same potential, but with implicit solvent, using the GBSA model, minimizing the structure to have a medium force coefficient of 6,4x10-8J·mol-1·cm-1which ensures a good local minimum. Of the trajectories simulated from 6 lower frequencies was found a relationship with the movements observed in molecular dynamics and expected the allosteric transition between the two crystal structures. Finally was calculate the temperature factor of the three trajectories of molecular dynamics, observing their temperature factors increase with increasing temperature, contrary to expectations of crystallography which decrease with the increase of the system temperature.

A Thermotoga marítima (Tm) é uma bactéria que vive em temperaturas na faixa dos 65 até 90°C, com temperatura ótima do redor dos 80°C. A proteína TM1030 de Tm, é um regulador transcricional da família TetR (Tetracycline repressor protein) reguladores da expressão génica das proteínas TetA e TetB (Tetracycline resistance protein). Neste trabalho se rodarem 200ns de trajetória de dinâmica molecular a três temperaturas (293, 323 e 353K) da proteína TM1030 (PDB-1Z77) usando o pacote GROMACS com o potencial Amber99 e solvente explicito numa caixa cúbica com 90Å de comprimento, observando que RMSD da estrutura média da trajetória é menor em relação à estrutura cristalográfica, além disso que num primer momento esse RMSD tem uma mudança grande e que se estabiliza com uma maior velocidade nas maiores temperaturas. Também foi feito um analise de modos normais na mesma estrutura usando o mesmo potencial, mas com solvente implícito, usando o modelo GBSA, minimizando a estrutura até ter um coeficiente de força média de 6,4x10-8J·mol-1·cm-1 que assegura um bom mínimo local. Das trajetórias simuladas a partir das 6 menores frequências se achou uma relação com os movimentos observados nas dinâmicas moleculares e os esperados na transição alostérica entre as duas estruturas cristalográficas. Finalmente se calculam os fatores de temperatura das três trajetórias de dinâmica molecular, observando que seus esses fatores de temperatura aumentam com o aumento da temperatura, contrario do esperado da cristalografia onde diminuam com o aumento da temperatura do sistema. / The Thermotoga maritima (Tm) is a bacterium who can lives at temperatures of 65 to 90°C, with optimum temperature around of 80°C. The TM1030 protein of Tm is a transcriptional regulator from TetR family (Tetracycline repressor protein) regulators of gene expression of the TetA and TetB protein (Tetracycline resistance protein). In this work 200ns of molecular dynamics trajectory was run at three temperatures (293, 323 and 353K) of TM1030 protein (PDB-1Z77) using GROMACS package with Amber99 potential and explicit solvent in a cubic box with length 90A, noting that RMSD of the average structure of the trajectory is smaller with respect to the crystallographic structure, in addition, in a first time this RMSD have a large change and stabilizes at a higher speed at higher temperatures. There was also an analysis of normal modes on the same structure using the same potential, but with implicit solvent, using the GBSA model, minimizing the structure to have a medium force coefficient of 6,4x10-8J·mol-1·cm-1which ensures a good local minimum. Of the trajectories simulated from 6 lower frequencies was found a relationship with the movements observed in molecular dynamics and expected the allosteric transition between the two crystal structures. Finally was calculate the temperature factor of the three trajectories of molecular dynamics, observing their temperature factors increase with increasing temperature, contrary to expectations of crystallography which decrease with the increase of the system temperature.