• Folding-proteico
• MD-simulations
• molecole-singole
• pinze-ottiche
• proteina-ligando

Questa tesi di dottorato riporta studi sul ripiegamento (folding) proteico e sull’interazione tra proteina e ligando effettuati con pinze ottiche e simulazioni di dinamica molecolare. Gli obiettivi principali di questo lavoro sono stati: i) elucidare i meccanismi che regolano il processo di ripiegatura della proteina HIV-1-proteasi, e ii) caratterizzare l'effetto del ligando octanoyl-CoA sul profilo energetico della proteina Acyl-CoA binding protein (ACBP). La tesi è articolata in cinque capitoli. Il capitolo 1 elucida i principi di base della spettroscopia di forza e descrive alcuni modelli teorici utilizzati per analizzare ed interpretare dati sperimentali ottenuti tramite manipolazione meccanica di singole molecole. Questo capitolo inoltre riporta alcuni risultati sperimentali recentemente pubblicati riguardanti il meccanismo di ripiegamento di alcune proteine e l’interazione tra proteine e ligandi. Il capitolo 2 fornisce, nella sua prima parte, una breve introduzione sulle pinze ottiche e sul loro meccanismo di funzionamento. Inoltre descrive le caratteristiche salienti del setup pinze ottiche costruito presso l'Università di Modena e Reggio Emilia. Nella seconda parte di questo capitolo, vengono descritte brevemente le proprietà strutturali e funzionali delle due proteine utilizzate in questi studi: HIV-1-proteasi e Acyl-CoA binding protein (ACBP). Il capitolo 3 riporta i risultati sperimentali ottenuti manipolando singole molecole della proteina HIV-1-proteasi con pinze ottiche ed risultati di simulazioni di dinamica molecolare che forniscono dettagli atomistici delle reazioni di denaturazione osservate sperimentalmente. Il capitolo 4 descrive gli studi di singola molecola eseguiti con pinze ottiche e simulazioni di dinamica molecolare mirati ad elucidare il meccanismo attraverso il quale l’octanoyl-CoA modifica la cinetica di denaturazione e ripiegamento della proteina ACBP. Il capitolo 5 riassume le conclusioni principali degli studi riportati in questa tesi.

This thesis reports the application of dual-beam optical tweezers and molecular dynamics simulation to the study of protein folding and protein-ligand interactions. The focus of this dissertation was to understand the complex folding behavior of HIV-1-protease and to study the effect of ligand binding on mechanical properties of Acyl-CoA-binding protein. The thesis is organized as follows. Chapter 1 explains the basic principles of force spectroscopy techniques, with a focus on optical tweezers, describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. It also highlights on some recent and exciting results that have emerged from single-molecule research field on protein folding and protein-ligand interactions. Chapter 2 gives the brief introduction of optical tweezers, its working principle and dual-beam optical tweezers setup at the University of Modena and Reggio Emilia. In the second part of this chapter, there is a brief description of model systems that I have used for my doctoral studies. Retroviral protease from human immunodeficiency virus type 1 (HIV-1-PR) is essential for the maturation of the virus and therefore, it has been identified as potential targets for anti-AIDS therapies. Since it plays an essential role in the life cycle of the HIV virus, many drugs has been designed so far to inhibit the activity of HIV-1-PR, but all these attempts have failed. One possible reason for these failures could be a poor understanding of the molecular behavior of this protein. Folding mechanism of HIV-1-PR has already been studied by traditional bulk techniques. These studies had given the information of overall thermodynamics and kinetics of the process, but they failed to provide insight into the ensemble of folding routes that individual molecules follow to reach native state. To shed light on the molecular behavior of HIV-1-PR we decided to study unfolding and refolding processes of this protein using optical tweezers. With the help of molecular dynamics (MD) simulations, we gain the atomistic details of these processes. This study has been described in chapter 3. Acyl-CoA-binding protein (ACBP) is involved in various biochemical processes such as signal transduction, regulation of lipid and energy metabolism, gene regulation and many more. It is well-known that ACBP binds to the acyl CoA esters with a broad range of acyl-chain length (C4-C22). This ligand binding may affect the molecular processes of ACBP. Using OT and MD simulations, we have investigated the effects of ligand binding on unfolding and refolding processes of ACBP. Chapter 4 explains different methods such as force-ramp, force-jump we have used to extract kinetic parameters of ACBP. The results obtained from our studies were compared with the previous ACBP alone (without ligand) single-molecule study. Finally, chapter 5 summarizes the conclusions of this thesis.