Some Experimental Methods

TL;DR: In this article, the authors highlight a few of the experimental methods to help in the elucidation of the structure of the protein molecule in solution, including the X-ray diffraction technique.

Abstract: This chapter highlights a few of the experimental methods to help in the elucidation of the structure of the protein molecule in solution. The determination of the structure of myoglobin in the crystalline form by the X-ray diffraction technique represents a milestone in the field of protein structure. It is important to learn whether the structure of the molecule is the same in solution as it is in the crystal. The temperatures at which these structures are determined are usually not far removed from the transition temperature. Therefore, the protein has marginal stability, and its secondary and tertiary structure can easily be affected by small interactions that differ in solution and in the crystal. While a variety of solvent and ionic effects may give rise to shifts in the ultraviolet spectra of proteins, it is possible, by using auxiliary data such as the pH dependence of reversible denaturation to determine whether the effects are partially because of side-chain hydrogen bonding. While the pH dependence of reversible denaturation can be explained in terms of a model involving hydrogen bonds, this does not preclude the possibility of accounting for the phenomenon in terms of a model based on hydrophobic bonding, including electrostatic effects.