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Srinivas Annavarapu Venkateshwarloo
M.S., Pharmaceutical Sciences - 2003
University of Mississippi
Thesis Advisor: Vikas Nanda, Ph.D.
Graduate Program in Biochemistry and Molecular Biology
CABM Room 010
Tuesday, July 20, 2010
D-amino acids are rare in nature. They, however, play functionally significant roles when present. Incorporating D-amino acids in protein design provides access to structures inaccessible to homochiral molecules and may potentially provide resistance to degradation by proteases that are chirality-specific. An indirect estimate of D-amino acid propensities for helical positions was made through study of naturally occurring left-handed helices and turns in the Protein Data Bank. Glycine was the most frequently occurring amino acid within helical turns, while Glutamine had the highest propensity at the C’ location with a fold-increase of 3.6 over random expectation. Other residues favoring this location are serine and threonine. Examination of structures revealed the ability of polar sidechains to form a helix-capping network with backbone to backbone and sidechain to backbone hydrogen bonds at the carboxy terminus.
D-amino acids were computationally modeled in the Trp-cage mini-protein using rotamers favorable for helix capping interactions. Melting temperatures determined through circular dichroism experiments revealed an increase in melting temperature of 29 °C with D-glutamine and 23 °C with D-alanine. Gains were also made in ÄGU, with D-glutamine and D-alanine variants gaining 1.8 and 0.8 kcal/mol respectively. Differential scanning calorimetry experiments demonstrated an increase in melting temperature with D-alanine, D-glutamine, D-serine and D-asparagine, although folded and unfolded baselines were indistinct. Tryptophan fluorescence experiments revealed a slight blue shift in emission spectra with D-amino acids, indicating reduced solvent exposure of tryptophan-6. At an excitation wavelength of 295 nm, emission peak for the D-glutamine variant was at 360 nm versus 363 nm for wild type. Nuclear magnetic resonance generated models demonstrate the presence of sidechain to backbone interactions with low energy populations of D-glutamine variant.
Molecular dynamics simulations suggest the ability of D-alanine, D-glutamine and D-asparagine to maintain Trp-cage structure, while D-serine was shown to destabilize the C-terminus of the á-helix as well as the 310-helix. These simulations also indicate the ability of D-glutamine, D-asparagine and D-serine to form sidechain to backbone hydrogen bonds at the carboxy terminus.
These results suggest that D-amino acid propensities may be used in the rational design of proteins. D-amino acids can stabilize the carboxy terminus of á-helices through reduction of backbone entropy and through tertiary sidechain to backbone interactions. The Trp-cage, stabilized using D-amino acids may be used as a scaffold for the development of novel biologically active molecules.