Question

Dealing with the stability of a protein i found in lab that the basic pH, the protein folded based on my results. However, how can it destabilize a proteins structure? Does it have to do with the temperature as well in the high pH?

Answer 1

Effect of pH and temperature-

In crystallizing a protein is to gradually force the macromolecule from solution by decreasing its solubility (that is, on increasing its supersaturation). Many factors can influence protein solubility, which depends on the protein’s surface charge. A protein’s solubility is usually quite sensitive to pH and to temperature as well in many cases.Hence optimal pH target for crystallization of a protein should be its isoelectric point. Because at its isoelectronic point, a macromolecule carries an equal number of positive and negative charges and is therefore electrostatically neutral. This would seem to be the best situation for mutually attractive electrostatic interactions.

In addition most macromolecules do have a pronounced solubility minimum at their pI values and some even precipitate at their pI. The outcome revealed that no correlation between the pI of crystalline proteins and the pH at which they were crystallized existed. The pI even appears inclined toward formation of amorphous precipitate over the crystalline state. Rather, most proteins were successfully crystallized near their physiological pH. Therefore, at present, the seemingly best approach is to crystallize a protein from a solution near its physiological pH. This has as advantage that the risk of denaturation (i.e. the unfolding of the protein under conditions where it is no longer stable) is it was explained that the temperature dependence of protein solubility is determined by the temperature dependence of the protonation and deprotonation reaction constants of the amino acid side chains in the protein structure. In spite of the fact that most proteins display a clear solubility dependence on temperature, this parameter is not very often used to control supersaturation, but is in the majority of the cases kept constant during the entire experiment. Crystallization has been reported to occur over the entire range from 0 to 40°C and in some cases even 60°C, although it is usually conducted at either 4°C or at room temperature, 25°C. Just as is the case with the pH, extremes in temperature tend to cause denaturation of proteins.

The aromatic amino acid Trp, Phe, and Tyr absorb UV light. After excitation, the electrons decay to the ground state through several processes. Some vibrational relaxation occurs, bringing the electrons to lower vibrational energy levels. Some of the electrons can then fall to various vibrational levels at lower principle energy states through a radiative process. The photons emitted are lower in energy and hence longer in wavelength. The emitted light is termed fluorescence. The wavelength of maximum fluorescent intensity and the lifetime of the fluorescence decay is very sensitive to the environment of the amino acids. Hence fluorescence can also be used to measure changes in protein conformation