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Myosins are molecular motor proteins involved in cell movement, vesicle and organelle transport by moving along the cytoskeletal actin filaments. They include a myosin heavy chain and at least one myosin light chain (LC). The latter are typically bilobal proteins like calmodulin, where each lobe comprises a pair of EF-hand Ca2+-binding motifs. The LCs bind to ~25-residue IQ motifs that loosely conform to an IQXXXRGXXXR consensus sequence, and impart rigidity that is crucial for myosin function. The highly motile amoeba Dictyostelium discoideum expresses seven class I myosins, two of which (MyoD and MyoB) recruit the specific LCs MlcD and MlcB, with MlcB being the first observed single-lobe LC. However, the LCs for the remaining D. discoideum class I myosins are unknown. Identifying and characterizing these LCs is one focus of this thesis, with an overall goal of understanding their role in myosin function and regulation. Nuclear magnetic resonance spectroscopy, site-directed mutagenesis, and computational modeling were used to determine the solution structure of apo-MlcB and identify the MyoB IQ motif-binding site. Apo-MlcB differs from the typical closed conformation of an EF-hand Ca2+-binding protein in the apo-state as helix 1 in its structure is splayed from the remaining helices. The MyoB IQ motif-binding surface is not altered by Ca2+, involves residues from helices 1 and 4, and from residues in the N-terminal canonical EF-hand Ca2+-binding loop, and represents a unique mode of IQ recognition by a myosin LC. Calmodulin was identified as the LC for MyoA and MyoE while another single-lobe LC, MlcC, bound to two of three IQ motifs in MyoC. The solution structure of MlcC was more similar to the C-terminal lobe of apo-calmodulin than to apo-MlcB. Chemical shift perturbation studies suggest that like apo-CaM, MlcC undergoes a global MyoC IQ motif-induced conformational change. Computational modeling of the MlcC-MyoC IQ complex indicates that this is a feasible mode of IQ recognition. The structures of MlcB and MlcC, with their different modes of IQ motif binding, provide novel insights into IQ motif binding specificity and begin to illustrate their role in myosin function and regulation. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-29 11:42:03.428

<p>Endozepine is an alternative name for the testis-specific isoform of the acyl-CoA binding protein (t-ACBP). Acyl-CoA binding proteins form a highly conserved family of proteins, which bind long chain fatty acid esters with nanomolar affinity. They are also known to be endogenous ligands to the --amino butyric acid (GABA) receptor in the central nervous system and to play a role in a wide variety of cellular functions such as vesicular trafficking, fatty acid biosynthesis and gene regulation. A role for endozepine in apoptosis was suggested through promoter gene trapping studies using CHO22 cells in which 90 % reduction in the expression of endozepine correlated with delayed mitochondrial permeabilization, a reduced activation of caspase-3 (an activator of apoptosis) and a consequent resistance to C2-ceramide induced apoptosis.</p> <p>Transduction studies using Tat-GFP-ELP fusion protein showed that endozepine restored the sensitivity of mutant CHO22 cells to C2-ceramide induced apoptosis. In this thesis, we have investigated two hypotheses for the involvement of endozepine in ceramide-induced apoptosis. The first hypothesis is that endozepine contributes to apoptosis through the transport of palmitoyl-CoA, a substrate required for the de novo synthesis of ceramide. The second hypothesis is that endozepine interacts directly with ceramide leading to interaction with peripheral benzodiazepine receptor and a subsequent opening of the mitochondria permeability transition pore, leading to apoptosis.</p>

A variable temperature 1H, 13C, and 6Li NMR study of n-propyl-6Li-lithium showed five different aggregates, similar to that in the literature as (RLi)n, n= 6, 8, 9, 9, 9. There were also a number of additional new species, identified as lithium hydride containing aggregates. Unexpectedly, a series of 13C{1H} 1-D NMR experiments with selective 6Li decoupling showed evidence for 13C-6Li spin-spin coupling between the previously reported (RLi)n aggregates and various hydride species.

We have reacted several derivatives of the anticancer drug cisplatin with N-acetyl-Lcysteine (N-AcCys) and N-acetyl-L-methionine (N-AcMet), which are two of the primary amino acid targets of platinum. NMR spectroscopy was used to monitor the reactions and determine the effect the different ligands would have on the platinum reactivity. Several of the platinum compounds were tested at pH of 4 and 7, and with platinum:amino acid ratios of 1:1, 2:1 and 1:2. Competition reactions between cysteine and methionine were done to confirm which would react with the platinum compound first. [Pt(dien)(NO3)]+ reacts faster with methionine than with cysteine at both pH 4 and 7 at a 1:1:1 ratio. [Pt(N,N,N',N',N"-pentamethyldiethylenetriamine)(NO3)]+ reacts with methionine faster at pH 4 but with cysteine faster at pH 7. This is most likely due to the thiol in the cysteine starting to deprotonate around pH 7. [Pt(Me4en)(NO3)2] (Me4en = N,N,N',N'-tetramethylethylenediamine) forms several products with N-AcCys at both pH 4 and 7, with the amounts of the products varying depending on the ratio of platinum and Cys. Mass spectrometry indicated one product as {[Pt(Me4en)(H2O)]2(N-AcCys)}2+, with two platinum compounds coordinated to a single cysteine. Lastly Pt[(en)(NO3)2] when reacting with N-AcCys at a ratio of 1:1 will coordinate with 2 different Cys molecules. With an excess of Pt the complex prefers to bind to only 1 Cys.