Calbindin D9k is a calcium binding protein that contains a helix-loop-helix motif, called EF-Hand. Members of the EF-Hand family are involved in calcium uptake and regulation of functions within the neuroendocrine and muscular systems. As calcium is involved in many cellular processes, such as muscle contraction and nucleotide metabolism, the function of these calcium-binding proteins is of great biological significance. Nuclear magnetic resonance spectroscopy (NMR) can provide a measure of protein dynamics, such as order parameters, that describe the residue-level mobility of a protein, including that of the backbone amide groups. These order parameters are often translated into conformational entropies, enabling an assessment of the entropic changes associated with unfolding or binding events. NMR studies that probe the stability and cooperative binding of calbindin D9k provide evidence that a temperature dependence of order parameters can identify regions of the protein that exhibit linear or nonlinear stability as a function of temperature. I will use molecular dynamics simulations to characterize the dynamics of Calbindin D9k and provide atomic level detail of the protein’s temperature dependent behavior over a temperature range of 3 to 63 Celsius. I will be comparing order parameters calculated from the simulations with experimentally derived order parameters for both the calcium loaded and unloaded states to determine the effects the calcium ions have on protein stability and conformation. Residues instrumental in calcium binding and structural stability are expected to exhibit smaller reductions in order parameters while non-structural regions should exhibit larger and more erratic reductions through increasing temperature. This study will further our knowledge of the dynamics of calcium binding proteins and offer an estimation of the free energy contribution that conformational entropy provides ligand binding. It may also be possible to extend these results to other calcium binding proteins known to act in disease and calcium deficiencies.

Proteins are functionally dependent on their 3-D structure. Many proteins share similar structural folds arising from either convergent evolution or a common ancestor. Understanding a protein’s folding pathway is a fundamental problem of biochemistry. The Daggett Lab created the Consensus Domain Dictionary to categorize common 3-D protein folds, termed fold families, based on various arrangements of secondary protein structure. Evidence suggests there may be common mechanisms in the folding and unfolding of members of a fold family. Here, we look for such characteristic mechanisms within 14 members of the Ubiquitin-like family, the 8th most commonly found protein fold. These proteins have a common core structure with sequence identities ranging from approximately 25% to 85%. Using Molecular Dynamics simulations to model the proteins denaturing at 498K, we compare transition states in the unfolding pathway, various atom-atom contact data, and the alpha-helical content amongst protein fold family members to analytically determine the existence of family-specific folding mechanisms. We anticipate that the early pathway will revolve around helix-turn-helix rotations or splays, sequential alpha-helical melting, and spatial separation of the four main beta sheets.

Proteins that share similar 3D structures, or folds, can be grouped together into fold families, and evidence suggests that there may be common folding mechanisms that exist within these fold families. Finding these common folding mechanisms will shed light on the way in which folding is determined among proteins, which is a major question in biochemistry. Here I investigate the dynamics and unfolding pathways of 18 members selected from the sixth most populated fold family, the SH3-like barrel domain. The SH3-like barrel fold is an all-beta fold, constructed of beta strands only. Using molecular dynamic simulations at high temperatures, the unfolding pathways, including the characterization of transition states, can be studied for each of these proteins. A broad analysis of native and non-native contacts and secondary structure content will allow common folding patterns to be determined across the fold family. It is anticipated that a hierarchical loss of contacts between the beta-strand of the fold, with those between the turns being maintained longer will be observed. Here I report an initial analysis of the unfolding of these 18 members of the SH3-like barrel fold family and present the observed commonalities, as well as variances across the pathways.