Biochemistry Online: An Approach Based on Chemical Logic

CHAPTER 2 - PROTEIN STRUCTURE

F: THERMODYNAMICS AND IMFs IN PROTEIN STABILITY

BIOCHEMISTRY - DR. JAKUBOWSKI

Last Update: 3/2/16

Learning Goals/Objectives for Chapter 2F: After class and this reading, students will be able to ...

Differentiate between general charge and specific ion-ion pairs and summarize their role in protein stability
Draw the structure of N-methylacetamide (NMA) and explain why it is a useful small molecule model to study the role of H bonds in protein stability
Draw a thermodynamics cycle for the transfer of a hydrogen bonded dimer of NMA from water to a nonpolar environment. From the DG0 for steps in the cycle, and extending this model to protein, predict if buried H bond formation drives protein folding
Explain if studies of low temperature protein denaturation, high temperature protein, and DGo transfer of nonpolar side chains from water to more nonpolar solvents support the hydrophobic effect in protein stability
summarize the relationship between the empirical Hofmeister series and preferential binding of reagents into the hydration sphere of protein to explain the effects of denaturants (urea, guanidine salts) and stabilizers (glycerol, ammonium sulfate) on proteins
Using benzene solubility in water as a model to study the role of hydrophobic effect in protein unfolding and by inference in protein stability, interpret graphs of DG0, DH0, DS0 and DCp for the transfer of benzene to water, as a function of temperature.
from the above graph, explain if trends in the thermodynamic parameters for benzene transfer into water predict the observed protein unfolding/stability behavior of proteins as a function of temperature?
Give a molecular interpretation of the observed DCp for the transfer of nonpolar molecules into water.
Describe chain conformational entropy, relate it to conformational changes in acyl side chains in single and double chain amphiphiles with temperatures, and describe it role in protein stability.
state which of several given explanations for the observed destabilizing effects of Asn to Ala mutations in protein account for those observation
summarize graphically the magnitude and direction of the major contributors (inter- and intramolecular forces and effects) to protein stability

F2. Ion - Ion Interactions

These could be investigated by altering pH or ionic strength. Why is that?

a. General Charge Interactions - Proteins denature at extremes of pH. At these extremes, proteins have a maximal positive or negative charge, as evident in graphs which show denaturation temperature vs pH for proteins.

Figure: Denaturation temperature vs pH for proteins

Under extremes of pH (but not so great as to catalyze peptide bond cleavage, electrostatic repulsions would cause the protein to denature. The folded, compact state has an increasing charge density at pH extremes, which could be alleviated by unfolding to a less dense state. But what about specific charge pair interactions? In contrast to the general charge interactions, these might actually stabilize a protein. Are they the predominant factor that determines stability?

b. Specific Charge Interactions (charge pairs) - If ion pairs are the source of protein stability, you would expect that high salt could disrupt them, and lead to denaturation. Although some salts do denature proteins, other stabilize them. Other evidence argues against this idea. Ion pairs are not conserved in evolution. In addition, the number of ion pairs in proteins is small (approx. 5/150 residues, with one of those buried). Also, the stability of a protein shows little dependence on pH or salt concentration (at low concentrations) near the isoelectric point, the pH at which proteins have a net zero charge.

The overall charge state affects not only the stability of a protein but also its solubility. Proteins are most insoluble at their isoelectric point since at that pH (where they have a net 0 charge) the proteins experience the least electrostatic repulsion and are most likely to aggregate and precipitate. Low salt concentration also promotes insolubility. Mutagenesis studies show that solubility can be increased by replacing nonpolar groups on the surface with polar ones. Pace (2009) cites studies on RNase Sa which has a maximally exposed Thr 76. If it is replaced with Asp, the solubility increased to 43 mg/ml but if it is replaced with Trp, it decreased to 3.6 mg/ml. His, Asn, Thr and Gln have a negative effect on solubility near the pI compared to Ala, a surprising result. Similar results were obtained compared to Ala when Arg and Lys were used. Smaller side chains, Asp and Ser, at position 76 increased the solubility over Ala.

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