Studies on conformational Transitions : Helix coil Transition Parameters for L-Alanine & L-Leucine in Mixed organic solvent system.

Abstract

The helix, proposed by Linus Pauling in the early 1950s, is known to be an important secondary structure in globular and fibrous proteins. It is of great interest to understand the propensities of individual amino acid residues to adopt the helical conformation. This information can be obtained quantitatively from studies of the reversible helix–coil transition in synthetic polypeptides. This transition can be induced by changing external variables such as temperature and solvent composition, and can be monitored by various spectroscopic techniques. The helix–coil transition involves nucleation and propagation of the helix, and has been treated adequately using statistical thermodynamic theories, such as the Zimm–Bragg model. The evaluation of the nucleation ( ) and propagation (s) parameters for naturally occurring amino acid residues provides a quantitative basis for comparing their relative stabilities in the helical state. Apart from their intrinsic value in clarifying the interactions that stabilize the helix, the and s parameters have important applications in understanding protein denaturation and in predicting helical regions in globular proteins. The helix–coil parameters and s have been determined for several naturally occurring amino acids (and a few others) in water. A particularly valuable technique is the “host–guest” method, wherein the transition parameters for a chosen “guest” residue are obtained from the helix–coil transition of random copolymers containing that residue and a “host” residue with known parameters. While aqueous phase helix–coil parameters are extremely useful, it is also important to assess how non aqueous solvents affect helical stability-particularly because the interior of globular proteins is relatively non polar. However, such parameters in non aqueous systems have not been available. This thesis attempts to fill this gap by determining these parameters for L alanine (Ala) and L leucine (Leu) residues in a solvent mixture containing 1,2 dichloroethane (DCE) and dichloroacetic acid (DCA). Random copolymers of these residues with benzyl L glutamate (BLG) (the host residue) in 82 wt% DCA were synthesized and their transition behaviour was analysed using copolymer theories. ________________________________________ Chapter I - Aim and Scope Chapter I describes the motivation, aim, and scope of the study, as summarized above. ________________________________________ Chapter II - Statistical Thermodynamics of the Helix–Coil Transition A brief survey of statistical thermodynamic theories of helix–coil transitions in homo and copolypeptides is presented. Particular attention is given to: • the Zimm–Bragg theory, • the nearest neighbour Ising model, and • the matrix formalism used to construct partition functions. Copolymer theories of Lifson, Allegra, and Lehman–MacTague-later used for analysis-are described. The chapter concludes with a survey of experimental helix–coil transition studies in both aqueous and non aqueous solvents. ________________________________________ Chapter III - Synthesis and Characterisation of Copolymers Chapter III describes the synthesis and characterization of random copolymers used in the study. Using N carboxyanhydrides of Ala and Leu, copolymers of type: • P(BLG –L Ala ) • P(BLG –L Leu ) were synthesized with small guest residue fractions to minimize complications. The copolymers were: • fractionated to reduce chain length heterogeneity, • analysed for composition (amino acid analysis), • analysed for molecular weight (sedimentation and viscosity), and • examined for conformation using ORD, circular dichroism, and infrared spectroscopy. ________________________________________ Chapter IV - Experimental Helix–Coil Transition Data The helix–coil transitions were induced by: • temperature variation, and • change in solvent composition, and monitored using the Moffitt–Yang parameter (b ) from ORD spectra. The transition curves (fractional helicity vs. temperature or solvent composition) were checked for: • reproducibility, • reversibility, and • concentration independence. Both Ala and Leu showed greater helical stability than BLG in the mixed organic solvent system, with Ala being the stronger helix former. ________________________________________ Chapter V - Analysis and Determination of and s Parameters This chapter presents the detailed analysis of the thermal transition curves using copolymer theories. The Zimm–Bragg and s parameters for the guest residues were determined by fitting experimental data to theoretical curves. Key findings: • was effectively temperature independent within experimental error. • The temperature dependence of s for Ala and Leu was obtained. • Enthalpy ( H°) and entropy ( S°) changes for the helix–coil transition were calculated from s(T). ________________________________________ Chapter VI - Discussion of Results Chapter VI discusses: • randomness of the copolymer sequences, • methods of fractional helicity determination, • applicability of copolymer theories, and • interpretation of , s, H°, and S° values. A major finding is that: • Ala has a larger s value than Leu in the non aqueous solvent, • but the reverse is true in water. This reversal is explained using stereochemical and energetic arguments, reflecting the influence of solvent environment. The implications for helix formation in globular proteins are discussed, emphasizing that amino acid helical stabilities depend strongly on their local solvent exposure.