UNDERSTANDING THE MOTILITY OF MOLECULAR MOTORS USING THEORY AND SIMULATIONS

Molecular motors are indispensable machines that are in charge of transporting cargoes within living cells. Despite recent advances in the study of these molecules, there is much that we still do not understand regarding the underlying mechanisms that allow them to efficiently move cargoes along polar cellular filaments. In this thesis, I report my investigation on two motor proteins superfamilies, dyneins and kinesins. Using theoretical modeling, we provide fundamental insight into their function. Dynein is a large motor that transports cargo along microtubules towards their negative pole. Unlike other motors, such as conventional kinesin, the motility of dynein is highly stochastic. We developed a novel theoretical approach, which reproduces a wide variety of its properties, including the unique step size distribution observed in experiments. Furthermore, our model enables us to derive several simple expressions that can be fitted to experiment, thus providing a physical interpretation. A less understood aspect of dynein is the complex set of allosteric transitions in response to ATP binding and hydrolysis, and microtubule binding. The resulting conformational transitions propel the motor forward to the minus end of the microtubule. Furthermore, its activity is regulated by external strain. Using coarse grained Brownian dynamics simulations, we show that a couple of insert loops in the AAA2, a sub domain in the AAA+ ring in the motor domain, play an important role in several of the alllosteric pathways. Kinesins are highly processive motor proteins that transport cargo along microtubules toward their positive poles. Experiments show that the kinesin motor domains propel the motor forward by passing each other in a hand-over-hand motion. However, there is a debate as to whether the motor domains do so in a symmetrical manner or an asymmetrical motion. Using coarse grained Brownian dynamics simulations of the kinesin dimer, we show that the kinesin stepping mechanism is influenced by the size of its cargo. Furthermore, we find that stepping occurs by a combinations of both the symmetric and asymmetric mechanisms. The results I present in this thesis are a testimony that theoretical approaches are invaluable to the study of molecular motors

Protein diversification through post-translational modifications, alternative splicing, and gene duplication

Proteins provide the basis for cellular function. Having multiple versions of the same protein within a single organism provides a way of regulating its activity or developing novel functions. Post-translational modifications of proteins, by means of adding/removing chemical groups to amino acids, allow for a well-regulated and controlled way of generating functionally distinct protein species. Alternative splicing is another method with which organisms possibly generate new isoforms. Additionally, gene duplication events throughout evolution generate multiple paralogs of the same genes, resulting in multiple versions of the same protein within an organism. In this review, we discuss recent advancements in the study of these three methods of protein diversification and provide illustrative examples of how they affect protein structure and function

How kinesin waits for ATP affects the nucleotide and load dependence of the stepping kinetics

Dimeric molecular motors walk on polar tracks by binding and hydrolyzing one ATP per step. Despite tremendous progress, the waiting state for ATP binding in the well-studied kinesin that walks on microtubule (MT), remains controversial. One experiment suggests that in the waiting state both heads are bound to the MT, while the other shows that ATP binds to the leading head after the partner head detaches. To discriminate between these two scenarios, we developed a theory to calculate accurately several experimentally measurable quantities as a function of ATP concentration and resistive force. In particular, we predict that measurement of the randomness parameter could discriminate between the two scenarios for the waiting state of kinesin, thereby resolving this standing controversy