Analysis of the secondary structures of the standard proteins myoglobin, hemoglobin, lysozyme and a-chymotrypsin by CD spectroscopy resulted in excellent agreement of estimated fractional composition with that observed by x-ray crystallography. CD spectroscopy was found to be very accurate for estimating a-helix and random coil content, but considerably less so for estimating b-sheet. Conversely, FTIR spectroscopic analysis of the same proteins proved to be more accurate for b-sheet estimation than either a-helix or random coil. Overall, CD spectroscopy was found to be superior to FTIR spectroscopy for the quantitative analysis of protein secondary structure, however, the two techniques are highly complimentary for protein structural studies. Both CD and FTIR methods were found to be useful for protein stability studies, where CD is suited to analysing a-helix stability through CD222 protein melts, and FTIR is capable of investigating protein aggregation phenomenon (together with 2D correlation spectroscopy) and b-sheet stability. The anhydrobiotic AavLEA1 nematode protein was discovered to be a natively unfolded protein with an extended tertiary conformation. From FTIR melt experiments this protein was shown to resist temperature-induced aggregation and to act synergistically with the trehalose disaccharide in retarding the aggregation of cytochrome c. AavLEA1 was also shown to be capable of producing protein fibrils by adopting a completely helical structure in the presence of high concentrations of TFE. Additionally, titration with Ca2+ resulted in an increase of ordered secondary structure and demonstrated AavLEA1’s ability to sequester cations. A second nematode anhydrobiotic protein (P. sup DJ-1) was found to be structurally very similar to the human DJ-1 homolog and exist as an a/b-mixed b- sandwich protein. Stability studies revealed that P. sup DJ-1 showed considerable temperature stability, both in terms of its helix domains and its resistance to aggregation. The structure of P. sup DJ-1 was found to be unaffected by high concentrations of H2O2, as was its temperature stability, leading to the conclusion that this protein functions as a molecular chaperone to relieve oxidative and/or heat stress, similar to current opinions on the functionality of the human homolog.[1] The Rab11-FIP3 homo-dimer predicted coiled coil protein fragment was confirmed by CD spectroscopy and TFE titration. For the Rab11-FIP2 homo-dimer, mutation of the valine residue 456 and the leucine residue 457 to glycine residues was found to destabilise the mutant relative to the wild-type. As such, these residues are concluded to be of central importance in the formation of the coiled-coil cap that protects the hydrophobic core from the aqueous environment. References: 1 Shendelman S, Jonason A, Martinat C, Leete T, Abeliovich A. DJ-1 is a redoxdependent molecular chaperone that inhibits alpha-synuclein aggregate formation. Plos Biology 2(11):1764-1773, 2004.