Small molecule binding and first hyperpolarizability of biomolecules probed by hyper-Rayleigh scattering in solution

Abstract

Application of spectroscopic techniques in studying the interaction between a large molecule such as a protein or DNA and a small molecule which serves a certain biological function has always been an area of much interest. Spectroscopic techniques allow the possibility of in situ non-destructive analysis of such intermolecular interactions, including binding between two molecules. Insight into the binding interaction is often crucial to understand and ascertain the role of small molecules in the biology of the cell. It is also important to investigate changes in the structure and properties of the biomolecules upon changing their environment, by altering the pH, temperature, salt concentration, and polarity of the environment. A variety of spectroscopic techniques is currently available to probe biomolecular interaction and binding. However, complex biological associations demand new tools to be invented and developed. The advent of lasers has provided many nonlinear optical techniques to be invented and developed with possible applications in biology.

In this work, hyper-Rayleigh scattering (HRS), or second harmonic light scattering in solution, a nonlinear optical technique, has been employed to probe various biomolecular interactions, which are new applications of the technique. Also, the molecular hyperpolarizability (?) of a large number of specially designed peptides and other small biologically important molecules has been investigated to probe the origin of quadratic nonlinearity in biological systems. In Chapter I, an introduction to the binding of small molecules to a large biomolecule and its importance in biology is presented. A general introduction to second-order nonlinear optics and various methods for the determination of quadratic molecular nonlinearity are provided in this chapter. The motivation and scope of the work done in this thesis are also outlined.

In Chapter II, the experimental set-up for the hyper-Rayleigh scattering (HRS) measurements at 1064 and 1907 nm is described. An outline of various other spectroscopies used in the present study, like UV/Visible, fluorescence, circular dichroism, and NMR, is given. This chapter also contains the procedure for the synthesis of several de novo designed peptides.

In Chapter III, the utility of HRS to determine binding constants has been illustrated. The systems investigated are: (i) pyridoxal phosphate, a cofactor of vitamin B6-dependent enzymes, with various amino acids, and (ii) all-trans retinal with n-butylamine and Gramicidin S. These binding processes proceed via Schiff’s base formation, and thus the interaction is covalent and strong in nature. In the experiment, one of the molecules was added to the other, and the second harmonic signal was monitored as a function of the addition of the titrant. The second harmonic titration data were analyzed using a nonlinear regression procedure for a single-site binding, and the equilibrium association constants were determined.

In Chapter IV, two binding constants of human serum albumin binding to bilirubin were obtained from second harmonic titration measurements. This is an example of binding of a small molecule to two non-interacting, independent sites of a protein. The titration curves were analyzed using a consecutive two-site binding mechanism. The values of the two association constants, K? and K?, determined by the HRS technique, are respectively 1.5 ± 0.43 × 10? M?¹ and 1.01 ± 0.16 × 10? M?¹, and the numbers agree well with the reported values obtained by other methods.

In Chapter V, the factors responsible for the first hyperpolarizability of polypeptides and proteins from their building blocks, that is, amino acids, are investigated. In a bottom-up approach, the first molecular hyperpolarizability (?) for amino acids, de novo designed peptides, and a few proteins was measured. The ? values have been correlated with the nature of the amino acid residues (aliphatic vs. aromatic), the length and the sequence of their appearance in the polypeptide chain, and its conformation.

In Chapter VI, the results on the measurement of first hyperpolarizability of all-trans retinal and related molecules are presented. The measurements using the external reference method resulted in realistic ? values, which are much lower than the previously reported values obtained using the internal reference method. The measured low ? values are also supported by calculations.

In the last Chapter VII, conclusions drawn from this work are presented, along with the future perspective in using HRS as a tool for biomolecular studies.