(i) Deoxyhypusine hydroxylase catalyses the final step in the essential modification of eIF5A. The enzyme was recently isolated and the gene sequenced in humans and yeast. Based on this sequence, the protein was predicted to contain several alpha helical repeats. Using circular dichroism (CD) measurements we showed that the protein is 77% helical with no detectable beta sheet. The secondary structure was not changed by removal of the catalytically important ferrous iron, nor by the mutation of conserved His or Glu residues involved in binding of the metal ions. (Kim Y.S. et al, 2006)? (ii) Resonance light scattering is an esoteric technique, which takes advantage of the complex behavior of the refractive index within the absorbance bands of high molecular aggregates of some chromophores. Recently numerous biochemical assays have been published, which claim to use this methodology. We have shown that most of the scattering peaks detected in these experiments are spurious, arising from the properties of the intrument's photomultiplier, not from the molecular species under study. A simple method for the detection of this artifact has been descibed. (McPhie P. 2006)? (iii) Antibody presentation is a key step in the initiation of the adaptive immune response, depending on the expression of costimulatory molecules. Immune-enhancing oligodeoxynucleotides (ODNs)increase the expression of these molecules. CD measurements were used to show that several of these regulatory ODNs contain guanine(G)-tetrad structures. Heat treatment of these ODNs which diminishes their regulatory properties also destroyed the G-tetrads. ((Wang J. et al 2007)? (iv) Optical studies on the structures of amyloid peptides and proteins are continuing.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2007
Total Cost
$319,125
Indirect Cost
City
State
Country
United States
Zip Code
Shewmaker, Frank; McGlinchey, Ryan P; Thurber, Kent R et al. (2009) The functional curli amyloid is not based on in-register parallel beta-sheet structure. J Biol Chem 284:25065-76
Fernandez, Cristina; Minton, Allen P (2009) Static light scattering from concentrated protein solutions II: experimental test of theory for protein mixtures and weakly self-associating proteins. Biophys J 96:1992-8
McGlinchey, Ryan P; Shewmaker, Frank; McPhie, Peter et al. (2009) The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis. Proc Natl Acad Sci U S A 106:13731-6
Phillips, Robert S; McPhie, Peter; Miles, Edith W et al. (2008) Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase. Arch Biochem Biophys 470:8-19
McPhie, Peter (2008) Concentration-independent estimation of protein secondary structure by circular dichroism: a comparison of methods. Anal Biochem 375:379-81
Wang, Jinhai; Roderiquez, Gregory; Jones, Taneishia et al. (2007) Control of in vitro immune responses by regulatory oligodeoxynucleotides through inhibition of pIII promoter directed expression of MHC class II transactivator in human primary monocytes. J Immunol 179:45-52
McPhie, Peter (2006) Resonance light scattering profiles must be corrected for instrument performance. Anal Biochem 348:157-9
Kim, Yeon Sook; Kang, Kee Ryeon; Wolff, Edith C et al. (2006) Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected]. J Biol Chem 281:13217-25