T e purpose of this review is to assess the nature and magnitudes of the dominant forces in protein folding. Since proteins are only marginally stable at room temperature,’ no type of molecular interaction is unimportant, and even small interactions can contribute significantly (positively or negatively) to stability (Alber, 1989a,b; Matthews, 1987a,b). However, the present review aims to identify only the largest forces that lead to the structural features of globular proteins: their extraordinary compactness, their core of nonpolar residues, and their considerable amounts of internal architecture. This review explores contributions to the free energy of folding arising from electrostatics (classical charge repulsions and ion pairing), hydrogen-bonding and van der Waals interactions, intrinsic propensities, and hydrophobic interactions. An earlier review by Kauzmann (1959) introduced the importance of hydrophobic interactions. His insights were particularly remarkable considering that he did not have the benefit of known protein structures, model studies, high-resolution calorimetry, mutational methods, or force-field or statistical mechanical results. The present review aims to provide a reassessment of the factors important for folding in light of current knowledge. Also considered here are the opposing forces, conformational entropy and electrostatics. The process of protein folding has been known for about 60 years. In 1902, Emil Fischer and Franz Hofmeister independently concluded that proteins were chains of covalently linked amino acids (Haschemeyer & Haschemeyer, 1973) but deeper understanding of protein structure and conformational change was hindered because of the difficulty in finding conditions for solubilization. Chick and Martin (191 1) were the first to discover the process of denaturation and to distinguish it from the process of aggregation. By 1925, the denaturation process was considered to be either hydrolysis of the peptide bond (Wu & Wu, 1925; Anson & Mirsky, 1925) or dehydration of the protein (Robertson, 1918). The view that protein denaturation was an unfolding process was

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Dominant forces in protein folding

A computer-assisted method for identifying protein sequences that fold into a known three-dimensional structure. The method determines three key features of each residue's environment within the structure: (1) the total area of the residue's side-chain that is buried by other protein atoms, inaccessible to solvent; (2) the fraction of the side-chain area that is covered by polar atoms (O, N) or water, and (3) the local secondary structure. Based on these parameters, each residue position is categorized into an environment class. In this manner, a three-dimensional protein structure is converted into a one-dimensional environment string. A 3D structure profile table is then created containing score values that represent the frequency of finding any of the 20 common amino acids structures at each position of the environment string. These frequencies are determined from a database of known protein structures and aligned sequences.

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Method to identify protein sequences that fold into a known three-dimensional structure

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.

https://science.sciencemag.org/content/sci/244/4908/1081.full.pdf

High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

Publisher Summary This chapter summarizes the experimental information on protein energetics. This field is developing fast and the concept of the energetics of protein structure has changed considerably during the past few years based on new findings. The proteins which are presented in the chapter are selected from a large number of proteins for which the thermodynamics of unfolding are studied in laboratory. The analysis of protein energetics presented in this chapter is based on several assumptions: (1) protein groups contribute additively, and proportionally as their surfaces, to the overall thermodynamic effects of unfolding; (2) the protein interior closely resembles an organic crystal in the way groups are packed and the energetics of the interactions among these groups are similar to those in the organic crystals; and (3) under certain conditions the denatured protein can be regarded as unfolded. The main criteria in choosing these proteins have been the reversibility of the denaturation process modeling unfolding, the completeness of this unfolding, the reliability of thermodynamic data on this process, and the resolution of the three dimensional structure of the given protein. The latter is important to investigate the correlation between thermodynamic and structural characteristics of protein, including the water-ASA of various groups in the native and unfolded states, the number of hydrogen bonds, and the extent of van der Waals contacts in the native state.

Energetics of protein structure.

Measurements of changes in structure and stability caused by 13 different substitutions for threonine 157 in phage T4 lysozyme show that the most stable lysozyme variants contain hydrogen bonds analogous to those in the wild-type enzyme and that structural adjustments allow the protein to be surprisingly tolerant of amino-acid substitutions.

Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme.

Crystallization of serine carboxypeptidases.

18 – Approaches toward the Design of Proteins of Enhanced Thermostability

The advent of directed mutagenesis has made it possible to alter protein structures at will. For the first time it is possible to design and to introduce modifications into a protein that are intended to change its behavior in predictable ways.

Crystallographic and Genetic Approaches Toward the Design of Proteins of Enhanced Thermostability

The lysozyme from bacteriophage T4 is being used as a model system to determine the factors that influence the folding and stability of proteins. The three-dimensional structure of the protein is known and lysozymes with modified properties arising from single amino acid substitutions have been obtained by classical selection techniques as well as by site-directed mutagenesis. By rationalizing the stabilities of the mutant proteins in terms of their observed three-dimensional structures we are attempting to quantitate the contributions that single amino acids make to protein stability. Our studies to date lead to the following conclusions. (1) Thermal stability is global. (2) Different types of interaction contribute to stability. (3) Changes in stability need not be associated with large structural changes. (4) An increase in the stability of an α-helix may enhance the stability of the protein as a whole. (5) Hydrogen bonds contribute to stability. (6) Protein structures can readily adjust to changes in amino acid sequence.

K. Wilson

Papers

Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme.

Crystallization of serine carboxypeptidases.

18 – Approaches toward the Design of Proteins of Enhanced Thermostability

Crystallographic and Genetic Approaches Toward the Design of Proteins of Enhanced Thermostability

Selected and Directed Mutants of T4 Phage Lysozyme