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

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Hydrogen bonding in globular proteins

Several sequence and structural factors have been proposed to contribute toward greater stability of thermophilic proteins. Here we present a statistical examination of structural and sequence parameters in representatives of 18 non-redundant families of thermophilic and mesophilic proteins. Our aim was to look for systematic differences among thermophilic and mesophilic proteins across the families. We observe that both thermophilic and mesophilic proteins have similar hydrophobicities, compactness, oligomeric states, polar and non-polar contribution to surface areas, main-chain and side-chain hydrogen bonds. Insertions/deletions and proline substitutions do not show consistent trends between the thermophilic and mesophilic members of the families. On the other hand, salt bridges and side chain-side chain hydrogen bonds increase in the majority of the thermophilic proteins. Additionally, comparisons of the sequences of the thermophile-mesophile homologous protein pairs indicate that Arg and Tyr are significantly more frequent, while Cys and Ser are less frequent in thermophilic proteins. Thermophiles both have a larger fraction of their residues in the alpha-helical conformation, and they avoid Pro in their alpha-helices to a greater extent than the mesophiles. These results indicate that thermostable proteins adapt dual strategies to withstand high temperatures. Our intention has been to explore factors contributing to the stability of proteins from thermophiles with respect to the melting temperatures (T(m)), the best descriptor of thermal stability. Unfortunately, T(m) values are available only for a few proteins in our high resolution dataset. Currently, this limits our ability to examine correlations in a meaningful way.

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Factors enhancing protein thermostability

Publisher Summary This chapter reviews the subject of noncovalent interactions in proteins with particular emphasis on the so-called weakly polar interactions. The chapter discusses the physical bases of the noncovalent electrostatic interactions that stabilize protein structure and the four types of weakly polar interactions that have been shown to occur in proteins are described with reference to some biologically significant examples of protein structure stabilization and protein–ligand binding. Atomic multipoles are estimated by fitting the atomic multipole expansion to the detailed features of the ground-state wave function obtained from ab initio quantum mechanical calculations. The amino acids phenylalanine, tyrosine, and tryptophan are traditionally grouped because their side chains are aromatic. The weakly polar nature of aromatic residues that leads to their interaction with oxygen and sulfur atoms suggests that they ought to be able to interact with themselves as well. Aromatic–aromatic interactions also play an important role in protein–ligand binding. The physical characteristics of noncovalent electrostatic interactions that stabilize protein structure have been described. In addition, an unusual group of weak electrostatic interactions in proteins, which have only recently been characterized, have been reviewed in detail and some examples of biological importance cited. These interactions, termed “weakly polar,” result from the characteristic distribution of partial charges in some amino acid side chain moieties and involve interactions among electronic monopole, dipole, and quadrupole moments.

Weakly Polar Interactions In Proteins

MOST enzymes are quickly inactivated above about 55 °C but those from thermophile bacteria are stable for long periods at higher temperatures1. We do not know why because so far their structures have proved too complex. For example although the tertiary and quaternary structures of the enzyme glyceraldehyde phosphate dehydrogenase from lobster muscle and from Bacterium stearothermophilus are alike their amino acid sequences differ by more than 130 out of some 330 positions which makes it hard to decide why the stearothermophilus enzyme is more stable. The electron transfer protein ferredoxin offers a better chance because its single polypeptide chain contains fewer than 60 residues; its structure is known and its heat stability and amino acid sequence have been determined in both mesophile and thermophile bacteria. We have built an atomic model of this protein, replaced its amino acid side chains in turn to correspond to the published sequences and searched for possible causes of the greater heat stability of ferredoxins from thermophile bacteria. We found that it arises mainly from external salt bridges linking residues near the amino terminus to others near the carboxy terminus. Haemoglobin A2 a minor fraction of adult human haemoglobin which is a little more heat stable than the major fraction, haemoglobin A, seemed another good choice because its amino acid sequence differs from that of A at only 10 positions. The atomic model suggests that at only two of these positions are the replacements likely to contribute to the extra stability of haemoglobin A2 one replacement providing an extra hydrogen bond between the α1 and β1 subunits and the other adding two non-polar interactions to a surface crevice within the β subunits. To account for the increased heat stability of the two proteins the extra bond energy provided by these interactions need not be larger than 10 kJ for ferredoxin or 5 kJ for haemoglobin A2.

H Raidt

Papers

Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2.