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Amino acid propensities in left-handed helical turns guide protein design using D-amino acid

by
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
Piscataway

Tuesday, July 20, 2010
3:00 pm


Abstract

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 GU, 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 -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 -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.


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