Conformational Properties of Polyglutamine Sequences Under Denaturing Conditions

Abstract

A number of diseases are caused by polyglutamine sequences in proteins such as Huntington's disease, spinocerebellar ataxia and many more. A better insight into the behavior of polyglutamine sequences is essential for understanding their role in diseases. Therefore, it is worth studying the behavior of polyglutamine sequences in one of the early steps of protein folding, i.e. loop formation. To this end, we are looking into the thermodynamics and kinetics of loop formation of polyglutamine sequences using engineered yeast iso-1-cytochrome c, which forms a loop between His(-2) and the heme in the denatured state. The His(-2) is part of a five amino acid N-terminal extension which is not found in eukaryotic cytochrome c and readily accepts homopolymeric inserts. All the inserts are between His(-2) and Ala(-1). Five variants were made in such a way that each variant contains 1 to 5 sets of the sequence QQQQQK. The lysine is present in order to maintain solubility. Stability of these variants was measured by CD spectroscopy and is compared with the previously reported polyalanine inserts [Tzul, F. O and Bowler, B. E. Proc. Natl. Acad. Sci. USA, 107, 11364-9 (2010)]. pH titration experiments were performed in 3 M and 6 M guanidine-HCl to measure equilibrium His-heme loop formation with all variants. The scaling exponents for loop formation are compared with those of polyalanine inserts in 3 M and 6 M guanidine-HCl, respectively. Kinetics of loop formation and loop breakage for polyglutamine variants were performed by pH jump methods in both 3 M and 6 M GdnHCl. The kinetics are compared with the previously reported data for polyalanine sequences and with data for His-heme loop formation in the denatured state of Rhodopseudomonas palustris cytochrome c'.