The energy profiles of atomic conformational transition intermediates of adenylate kinase

The elastic network interpolation (ENI)1 is a computationally efficient and physically realistic method to generate conformational transition intermediates between two forms of a given protein. However it can be asked whether these calculated conformations provide good representatives for these intermediates. In this study, we use ENI to generate conformational transition intermediates between the open form and the closed forms of adenylate kinase (AK). Based on Cα-only intermediates, we construct atomic intermediates by grafting all the atoms of known AK structures onto the Cα atoms and then perform CHARMM energy minimization to remove steric conflicts and optimize these intermediate structures. We compare the energy profiles for all intermediates from both the CHARMM force-field and from knowledge-based energy functions. We find that the CHARMM energies can successfully capture the two energy minima representing the open AK and closed AK forms, while the energies computed from the knowledge-based energy functions can detect the local energy minimum representing the closed AK form and show some general features of the transition pathway with a somewhat similar energy profile as the CHARMM energies. The combinatorial extension (CE) structural alignment2 and the k-means clustering algorithm are then used to show that known PDB structures closely resemble computed intermediates along the transition pathway

Infrared Measurements of Protein Conformational Dynamics

The topic of how a protein folds has been a major area of research for several decades; however, important details about this process are still undetermined. Experimental limitations in the study of protein folding are a result of no technique possessing both the necessary spatial and temporal resolution. This Thesis presents several studies conducted with the goal of expanding upon the experimentalist\u27s toolbox, involving new methods of interrogating and/or perturbing protein systems of interest. The early chapters of this Thesis describe our efforts using established synthetic methods to extend the utility of infrared spectroscopy in the study of protein folding. Specifically, we show that, using the strategy of cysteine alkylation, we can incorporate novel vibrational probes into proteins in a site-specific manner. We also show that combined sidechain mutagenesis, probing at multiple frequencies, and isotopic labeling to obtain secondary structural resolution in infrared studies of protein folding, in the process uncovering details about the folding mechanism of the Trp-cage miniprotein. Similarly, we illustrated the use of thioamides as site-specific reporters of backbone-backbone hydrogen bonding, and applied this functionalization to the Trpzip2 beta-hairpin system to validate its proposed folding mechanism. Further work involved using D-amino acids to interrogate turn regions in proteins, specifically examining Trp-cage folding. The later chapters of this Thesis are focused on the effects of extrinsic molecules on the structural ordering of proteins. Taking advantage of the lack of tertiary structure of intrinsically disordered proteins, we examined the effect that trifluoroethanol has on protein folding, and found evidence that this cosolvent acts as a nano-crowder. We also introduced the idea of using phototriggers to modify the free energy landscape of folding for a given protein, and demonstrated that a peptide that typically folds in an activated (barrier-containing) manner can be made to fold in a downhill fashion upon irradiation

Méthodes de simulations moléculaires accélérées : application et développement

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Crystallographic B-factors Highlight Energetic Frustration in Aldolase Folding

Kinetic Simulations of the folding and unfolding of the mammalian TIM barrel protein aldolase were conducted to determine if a minimalist monomeric G (o) over bar model, using the native structure to determine attractive energies in the protein chain, could capture the experimentally determined folding pathway. The folding order, that is, the order in which different secondary structures fold, between the G (o) over bar model simulations and that from hydrogen-deuterium exchange experiments, did not agree. To explain this discrepancy, two alternate variant of the basic G (o) over bar model were simulated: (1) a monomer G (o) over bar model with native contact energies weighted by a statistical potential (SP model) and (2) a monomer G (o) over bar model with native contact energies inversely weighted by crystallographic B factors (B model). The B model demonstrated the best agreement between simulation and experiments. The success of the B model is attributed to the ability of B factors to highlight local energetic frustration in the aldolase structure which results in weaker native contacts in these frustrated regions. The predictive success of the B model also reveals the potential use of B factor information for energetic weighting, in general protein modeling