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Mechanisms of protein folding:

The folding of a protein is the process by which an incorrect unfolded conformation of the protein folds to its correct natural shape. This biological process is important because incorrectly shaped (misfolded) proteins can sometimes be the trigger of human ailments as Alzheimer’s, the variant Creutzfeldt-Jakob “mad cow” disease and type II diabetes.

We perform atomically detailed simulations to understand the folding process of increasingly complex proteins. Normal molecular dynamics (MD) can not be used to study this process because the folding times are usually larger than a millisecond, well beyond the time range that can be studied with MD (one hundred nanosecond). Therefore novel approximate techniques are employed like the stochastic difference equation in length (SDEL) algorithm to study these non-equilibrium processes.

Determination of the folding mechanism of two proteins of the cold shock protein family

The cold shock proteins (Csp) are small single-domain proteins with a five-stranded antiparallel β-sheet structure. These are nucleic acid binding proteins that act as chaperones for RNA at low temperature to keep mRNAs free of secondary structure formation.

We used (SDEL) to compute more than 20 folding pathways for two members of the Csp family. SDEL is a boundary value algorithm, i.e. trajectories connecting two known conformations of the system are obtained. The two boundary states in this case are a complete unfolded conformation of the protein and the correct native structure. Our results show an early collapse of the protein in agreement to experimental data and offer some structural insights about the initial folding step of these proteins.

Attach two figures with their respective captions.

 Kinetics of cytochrome c folding: atomically detailed simulations

Cytochrome c is an important protein in the process of creating cellular energy in the mitochondria. Its folding process has been studied extensively in the past with several experimental techniques.  We used SDEL to compute 26 folding pathways for this protein starting from coiled-like conformations. The results of our simulations agree with two major experimental observations: (1) the two terminal helices form first than the middle helix; (2) the folding process start with an initial hydrophobic collapse of the protein follows by further collapse accompanied with secondary structure formation. We characterized structurally the molten globule state of this protein and proposed a role to non-bonded interactions during the folding process.

 Figure; Upper left panel: The number of hydrogen bonds of a given helix (N, C or the 60’s helix) as a function of the path length. Average over 26 trajectories is reported. Upper right panel: A contour plot summarizing the folding progress along two reactions coordinates: The radius of gyration and the number of residues in helical conformations. Experimental points were taken from Akiyama et al. (PNAS 2002, 99:1329). Lower panel: Ribbon view of Cyt C at four different positions along one of the folding trajectory. The amino acids of the N, C and 60’s helices are in red, purple and yellow, respectively, the heme group is in pink and the rest of the backbone chain is in cyan.

Larger single-domain proteins:

We will study the folding process of more complex proteins like apomyoglobin and β-lactoglobulin. Folding pathways for these proteins will be obtained using an explicit atomic treatment of the aqueous environment. Apomyoglobin folding has been studied experimentally by the group of Randy Larsen (here at USF) using photothermal techniques. These experiments probe changes in enthalpy and volume during the folding process. To extract volumetric information from the simulations, we can perform constant pressure and temperature MD simulations using conformations extracted from the SDEL trajectories. A pluck out procedure, designed by Brian Space (other colleague in the department) and Larsen provides these volume changes. Umbrella sampling and simplified continuum treatments can also be used to extract energetic information (free energies) for the folding of these proteins. Results of these computational studies will offer structural characterization of possible intermediates and for the β-lactoglobulin case

a possible elucidation of the nature of the non-native helical structure observed experimentally during its folding.

Multidomain proteins:

We will characterize the folding and assembly process of two-domain proteins (gene-3 protein of phage fd and the phosphoglycerate kinase). Understanding of the folding process will also offer clues about their function (the first involved in E. coli infection by the phage fd and the second is an important enzyme of the glycolytic pathway).

Insertion and folding of membrane bound proteins:

This more complex process because it involves the interaction of lipid molecules with the protein will be studied using a combination of SDEL with an implicit solvent/membrane environment.

Conformational changes of protein ion channels and enzymes

·        Studies of the gating mechanism of ion channels (i.e., the process of opening and closing of the channel) will be studied using SDEL with explicit treatment of water and lipid molecules. Channels to be studied are the KcsA K⁺ channel and the OmpA porin channel.

·        We will study the structural changes occurring during the activation-inactivation process of Src Kinases. These are important enzymes in the phosphorylation pathway of eukaryotic cells.

Cotranslational folding

Simplified models are used to study folding of proteins during the translation process occurring in the ribosome. The ultimate goal will be to study this process using simulations with atomic detail.