Lab 9: BACKGROUND
As you experienced with albumin, it is not very difficult to denature proteins. In a research setting, we want to denature proteins in a more controlled, quantitative way so that we can gain insight into the thermodynamics of protein unfolding, which can tell us about the stability of the protein. To quantitatively measure protein denaturation of HCAII we will take advantage of two biochemical phenomenon: chemical denaturation and fluorescence spectroscopy.
In the case of boiled egg whites, the proteins were denatured, but then formed larger bond networks that knitted many proteins together, causing the change in color and firmness. In contrast, when denaturing proteins in a research setting, chemical denaturants called chaotropes (ex. urea and guanidinium HCl) are used. These agents essentially outcompete for hydrogen bonding to water, thereby weakening the hydrophobic effect to the point that proteins no longer fold. These agents also prevent the formation of larger aggregates, resulting in fully denatured protein molecules floating freely in solution. In 551 lab, urea will be used to incrementally reduce the fraction of folded HCAII in a sample at room temperature, in such a way that the fraction of folded and unfolded proteins can be measured, enabling thermodynamic analysis of protein stability (Bennion and Daggett, 2003).
To measure how much folded and unfolded protein is in each sample, we will use fluorescence spectroscopy. This is a technique used to measure a fluorescent signal inherent to fluorescent molecules. When a fluorophore absorbs a specified wavelength of light, its electronic state is excited to a higher energy state. The higher state subsequently drops in electronic state, which coincides with emitting photons at a lower energy wavelength (Figure 9.2).
Fluorimeters can detect this emitted wavelength, which can then be used to infer various properties of the fluorophore and of its surroundings. This technique can be applied to study, for example, protein stability, enzymatic activity, and ligand binding. Advantages of this technique include the ability to use a wide range of experimental conditions, the requirement for a small amount of sample, and the ability to detect fast changes.
In this lab, we will exploit the fact that a fluorescent amino acid (tryptophan) emits fluorescence at a different wavelength depending on whether the protein is folded or unfolded. Because tryptophan is already present in protein, we refer to this phenomenon as “intrinsic fluorescence” (to differentiate from a case where a fluorophore has been added chemically to a protein). This intrinsic protein fluorescence due to tryptophan is observed when the protein is excited at 280nm and its emission is observed at a different wavelength (e.g. 310nm-370nm).
HCAII contains seven tryptophans; you examined them during Lab 1. Tryptophan fluorescence depends on the polarity of the environment surrounding the tryptophan and can therefore indi