Analysis and modulation of protein stability Angelo Fontana University of Padua, Padua, Italy Numerous site-directed mutagenesis experiments have provided new insights into the stabilizing role of the individual forces and interactions within a globular protein molecule. Some useful guidelines and procedures are now available for producing genetically more stable proteins. Examples are the introduction of disulfide bonds, ion-binding sites, salt bridges, hydrophobic residues or hyrogen bonds, and the improvement of hydrophobic packing or s-helix propensity. Moreover, it is now clearly recognized that thermophilic (and, in general, extremophilic) bacteria produce highly stable proteins and enzymes of practical interest. Current Opinion in Biotechnology 1991, 2:551-560

Introduction The objective of this review is to consider briefly the results of recent successful protein engineering experiments aimed at analyzing and enhancing the stability of proteins. This is an important area in fundamental research because, only with a quantitative understanding of the forces and interactions that determine the protein structure, will it be possible both to understand the mechanism of protein folding [i.,2.] and to design d e n o v o protein molecules with specific structure and function [3]. Moreover, the problem of protein stability is the subject of considerable research because protein lability hampers successful applications of enzymes in various fields of chemical technology, food industry, medicine and analytical chemistry. It must be emphasized that, in the interests of brevity, this is not intended to be an exhaustive review of the literature associated with the protein stability problem. Instead, a personal selection of issues will be considered and the relevant results of last year's literature commented upon. For a more complete coverage of the field, the reader is referred to several recent reviews [4~,9",10",11".].

Measuring protein stability The net stability of a globular protein is determined by the free energy difference between the native/folded and denatured/unfolded states. The measure of the free energy of stabilization of a protein is usually straightforward for small, single-domain proteins that obey a two-state model of reversible unfolding, although it can be more complex for larger, multidomain proteins. The conformational stability of almost all naturally occurring proteins is unexpectedly small (5-15kcalmo1-1 ) because the factors that determine the stability of the folded proteins are offset by those favoring the unfolded state [12,13,14-.]. Estimates of AG

can be obtained from both thermal and urea or guanadine hydrochloride denaturation curves, which are obtained following the protein-unfolding process using an appropriate structural probe, such as far- and near-ultraviolet circular dichroism for secondary and tertiary structure, respectively [15,16.]. Calorimetric techniques offer unique means of analyzing the thermodynamics of protein denaturation, as they allow a direct measure of the energetics of the Unfolding process [17]. The methods currently employed to estimate protein stability have been summarized recently [18",19"]. In the earlier literature, protein stability was often evaluated in terms of rate of protein denaturation. This involved subjecting a protein sample to either high temperatures or to the presence of a protein denaturant, and then testing for residual activity or precipitate formation. This type of analysis of protein stability depends on irreversible chemical and physical processes and involves kinetic as well as equilibrium aspects. Whereas reversible unfolding processes of proteins are very useful for obtaining thermodynamic parameters, in practice, protein stability is normally defined by resistance to irreversible processes. There is a relationship between these two parameters, however, as most irreversible damages of proteins (chemical modification, proteolysis, aggregation, adsorption) require prior protein unfolding. The correlation between reversible and irreversible denaturation of proteins is discussed in an excellent review [20].

Engineering protein stability A number of fundamental aspects of protein structure and stability have been addressed using protein model systems for which detailed structural and functional data were already available. In this respect, proteins such as bacteriophage T4 lysozyme [21..], staphylococcal nudease [22--24.], £-repressor [25"], ribonuclease T1 [26]

(~ Current Biology Ltd ISSN 0958-1669

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Protein engineering and a-amylase [27",28"] were favored models for examining in a systematic way the various factors thought to be important in determining protein stability. These numerous experimental studies allowed the analysis of the role and contribution of the different forces that stabilize proteins, namely, hydrophobic effects, hydrogen bonds, electrostatic and van der Waals interactions, the a-helix propensity, disulfide bridges and ion-binding (see below). Excellent accounts and up-to-date reviews on the genetic analysis of protein stability are available [4-8,11..]. Single-site mutations of proteins have proved to be powerful tools for analyzing the individual interactions within a globular protein. For example, the role of glycine in protein stability has been analyzed by replacing it with other residues. Glycine lacks a [3-carbon and has greater backbone configurational entropy than other branched amino acids (e.g. alanine) and so requires more free energy to transfer from the unfolded to the folded state. Thus, the replacement of glycine with alanine in T4 lysozyme [29], neutral protease from Bacillus stearothermophilus [30] and £-repressor [31], resulted in an increase in protein thermal stability by 0.4-0.8 kcal m o l - 1. As an alternative, it was proposed that the stabilizing effect of the glycine-to-alanine substitution was a result of the replacement within an a-helix of a poor helix-forming residue (glycine) with an efficient helix former (alanine) [30,31]. On the other hand, when several glycineto-alanine mutations were carried out on helices of the oligomeric enzyme glyceraldehyde-3-phosphate dehydrogenase, only one of them was found to stabilize the enzyme and it was proposed that the stabilizing effect was a result of an internal cavity in the native enzyme being filled [32"]. The results of several protein engineering studies indicate that a possible principle of protein stabilization involves increasing the intrinsic helical stability of individual helices. A valine-to-alanine (Va1131Ala) mutant of T4 lysozyme designed to reduce the strain within an a-helix and thereby increase the stability of the protein was found to be slightly more thermostable than the wildtype lysozyme [33"]. By analogous reasoning, protein stabilization can be achieved by the correct positioning of charged residues into helical segments. In fact, the macrodipole of helices in proteins appears to be stabilized by charged residues at helix termini, that is, by acidic and basic amino acids at amino- and carboxy-termini, respectively. Thus, Ser38Asp and Asn144Asp mutations at the amino-terminal ends of helices in T4 lysozyme enhance protein stability [34]. Finally, it is expected that stabilizing mutations can be produced by replacing in helical segments the helix-breaker proline. However, substitution of Pro86 in a helix in T4 lysozyme with residues of different helix propensity had little effect on protein stability, implying that it is actually the structural context of each amino acid residue that dictates its specific role in protein structure and stability [35]. Alanine appears to be the most stabilizing amino acid residue within an a-helix, as shown by the fact that short alanine-based peptides form unexpectedly sta-

ble a-helices in water [36"-38"]. In a recent study, Matthews and coworkers [39"'] introduced a series of alanines within the 126-134 residue a-helix of T4 lysozyme. In the wild-type lysozyme, this a-helix contains alanine residues at positions 129, 130 and 134. The mutant containing three additional alanines at positions 128, 131 and 132 has a melting temperature (T m) 3.3°C above the wild-type lysozyme. Other protein variants containing single additional alanines in this helix were also more stable, with the exception of the mutant prepared by replacing the buried residue Leu133 with alanine, suggesting that this residue is critical for the folding and stability of T4 lysozyme. Overall, these data support the idea that replacement of solventexposed residues within a-helices with alanine residues might provide a general method of protein stabilization. A general observation that has emerged from numerous experiments of site-directed mutagenesis of proteins is that many amino acid residues in a protein sequence can be replaced with little if any effect on protein function, folding and stability. This high variability of sequence is tolerated mostly at solvent-exposed or mobile sites in the folded protein, whereas replacements of residues located in the interior of the protein often lead to destabilization. The most detailed studies on the tolerance of an amino acid sequence to change have been conducted by Sauer and coworkers [8,40..]. A combinatorial cassette mutagenesis technique was used to generate a large number of substitutions in the amino-terminal domain of K-repressor. This helical domain consists of 92 residues and binds to operator DNA as a dimer, with dimerization mediated by hydrophobic packing of a-helix-5. Random mutagenesis of helix-5 (residues 75-91) allowed the production of many mutant proteins which were identified and selected on the basis of a functional test. It was shown that some positions could tolerate very few substitutions, whereas others accepted a great variety. The positions which do not easily tolerate changes tend to be those that are buried in the protein structure [40.-]. More recently, the mutagenesis approach for analyzing the functionally acceptable amino acid substitutions was extended to helix-1 (residues 8-23) of X-repressor. Again, the results demonstrated the severe limitations of the number and type of residues tolerated at buried positions (Fig. 1) [41"]. The results of these studies illustrate the degeneracy in the information content of the protein amino acid sequence for specifying a particular folded and functional globular protein, as well as that buried hydrophobic residues are most important in determining the conformation and stability of globular proteins. Indeed, a mutagenic study of interior residues in ~,-repressor revealed that the hydrophobicity of these residues is an important factor and that the precise identity of the hydrophobic residues is less important [42]. This is also evident from the analysis of homologous or related protein structures [43,44,45,]. Electrostatic interactions between charged groups in globular proteins play an important role in protein stability, but this role is heavily dependent upon their environment. Charged residues buried within the hydropho-

Analysis

bic core of a protein are destabilizing whereas, if they are exposed at the protein surface, they contribute little to protein stability [10-.,11.-,46]. Protein engineering experiments have been carried out to analyze the role and magnitude of electrostatic interactions in protein stability. A recent example is that of T4 lysozyme, in which a specific salt bridge Asp70--His31 contributes 3-5 kcalmol-1 to the free energy of the folded state [47"]. Mutation of either or both charged groups to neutral residues produced the same decrease of 11°C in Tm, indicating the existence of a specific electrostatic interaction. A salt bridge Asp12-Arg16 in an cx-helix on the surface of a bamase mutant increases the protein stability by 0.5 kcal mol-1. The wild-type bamase contains threonine instead of arginine at position 16 of the chain and thus lacks this electrostatic interaction [48.].

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Fontana

A quite common finding of numerous experiments on engineering protein structures is that the effects of sitespecific mutations on protein stability are additive. Deviation from simple additivity has been observed when the individual mutations strongly interact with one another by proximity effects. The additivity has been demonstrated with mutants of staphylococcal nuclease, the aminoterminal domain of ~.-repressor, t h e cz-subunit of E~ cherichia coli tryptophan synthetase, T4 lysozyme, and the gene V product of bacteriophage fl (see [49"] for references). The additivity of changes in free energy of stabilization implies that interactions within globular proteins are highly localized and that individual mutations do not grossly alter the protein structure. Although this may be true for protein mutants that stably fold into a rigid structure, it seems that protein stability arises from a sum

Arg

Arg Lys Asn Ser Thr Gly

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85

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88

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Fig. 1. Functionally acceptable residues in the helix-1 (top) and helix-5 (bottom) regions of the amino-terminal domain (residues 1-92; in bold) of H-repressor obtained by a combinatorial cassette mutagenesis technique. The protein binds to operator DNA as a dimer, with dimerization mediated by hydrophobic packing of ~-helix-5 of one monomer against 0~-helix-5' of the other monomer. There is a wide range in tolerance to amino acid substitutions in both helical regions of the protein. A strong correlation exists between the number of allowed residue exchanges at a given position and the fractional solvent accessibility of the residue at that position in the crystal structure. At surface positions, a number of different residues are allowed whereas, at buried positions, few exchanges are tolerated. Reproduced, with permission, from [41.-].

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Proteinengineering of local interactions, such that it can be engineered to any desired value by introducing multiple, substitutions Into a protein molecule. The non-covalent stabilizing effects would be expected to reach a plateau as, for example, denaturation at high temperature may be controlled by irreversible denaturation effects (such as deamidation at asparagine or glycine residues and [3-elimination at cysteine residues) [50].

Stabilization by disulfide bonds Because of the evidence that disulfide bonds (and other chemical crosslinks) play a prominent role in stabilizing extracellular proteins, the engineering of intramolecular disulfide bonds into globular proteins is an obvious strategy for the improvement of protein stability. The stabilizing effect of a disulfide bond is presumed to be of entropic character because the entropy of the unfolded protein decreases significantly and the difference between the free-energy of the native and the denatured protein therefore increases. To date, non-natural disulfides have been introduced by site-directed mutagenesis into a number of proteins, such as dihydrofolate reductase, phage T4 lysozyme, subtilisin and ~-repressor (see [51] for a review and references). The procedure proved to be successful for stabilizing proteins in a number of cases, although it failed in a few instances. This variable success results from the fact that additional disulfide bridges in a protein should be introduced in a manner that does not cause strains in the polypeptide chain that might lead to structural defects and instability [52]. The engineering of disulfide bonds into the disulfidefree polypeptide chain of phage T4 lysozyme has been the subject of intense and detailed investigations [53]. The crystal structure of a thermostable mutant of T4 lysozyme with a disulfide bond between amino acid positions 9 and 164 has been determined at 1.8A resolution [54°]. The wild-type and crosslinked lysozymes have similar overall structures and crystallographic temperature factors, indicating that the introduction of the disulfide bond does not impose rigidity on the folded protein structure. These data support the belief that disulfide bonds increase the stability of proteins by reducing the backbone configurational entropy of the unfolded state. In a recent study [55], mutants ofT4 lysozyme with multiple disulfide bridges crosslinking residues 3-97, 21-142 and 9-164 were prepared and their functional and stability properties investigated. Studies on the reversible thermal unfolding of the mutants revealed that the increase in Tm resulting from individual disulfide bonds is additive. The triple-disulfide mutant unfolds at a temperature 23.4°C higher than the wild-type enzyme. The study demonstrates that individual disulfide bridges can be combined to enhance protein stability dramatically (Fig. 2). A mutant form of ribonuclease T1 in which a third disulfide bond has been introduced between residues 24 and 84, besides the two disulfide bonds of the wild-type

species, maintained both secondary structure and nucleolytic activity at a higher temperature than the wild-type species [56"]. a disulfide bond was engineered in the cysteine-free subtilisin E from Bacillus subtilis by introducing by site-directed mutagenesis cysteine residues at positions 61 (wild-type; glycine) and 98 (wild-type; serine) of the chain [57"]. The disulfide mutant showed a Tm of 63°C, which was 4.5°C higher than that observed with the wild-type enzyme. Interestingly, the sites for cysteine mutations were chosen on the basis of the amino acid sequence of the sefine-protease aqualysin I from the extreme thermophile Tbermus aquaticusYT-1. Aqualysin I is homologous to subtilisin E and contains four cysteine residues forming two disulfide bonds, which seem to be responsible for the unusual thermostability of the enzyme. A computer method has been developed for predicting whether or not a cysteine residue participates in a disulfide bond in a protein [58,]. Because the amino acid residues flanking the cysteine residue greatly influence disulfide bond formation, it is possible to predict the location of disulfide bonds in a protein with high confidence. It is likely that this method will be used in protein engineering studies. Amino acid sequences can be searched for residues that appear to be good candidates for mutation into cysteine, on the basis of the surrounding amino acids having a high propensity for disulfide bond formation. Moreover, the method can be used to increase or decrease the stability of existing disulfide bonds through the alteration of the amino acid sequences surrounding the cysteine residues. Attempts to test these strategies experimentally are awaited with interest.

Stabilization by ion binding Proteins are often stabilized against thermal inactivation when heated in the presence of relatively high salt concentrations (0.2-2 M) [59], whereas in the presence of moderate salt concentrations, the stabilization of proteins results mainly from the relatively weak binding of cations and anions by the native, folded conformation of the protein [15]. The influence of molar concentrations of salt on the stability of soluble proteins is explained in terms of salting-out and salting-in effects. Ions with salting-out effects cause the apolar residues to be even more soluble than they are in water, and stabilize protein structures by inducing more clustering of hydrophobic groups within the globular protein. At the same time, however, apolar residues at the protein surface tend to interact intermolecularly, thus favoring protein aggregation and precipitation. Salting-in ions make apolar groups more water-soluble and thus destabilize proteins by creating an environment that favors their unfolding. The effects of neutral salts on both structure and conformational stability of proteins have been reviewed [60]. The structure and stability properties of proteins and enzymes from halophilic bacteria are in apparent contradic-

Analysis and modulation of protein stability Fontana

Fig. 2. (a) To investigate the effect of multiple disulfide bonds 6n protein stability, mutants of bacteriophage T4 lysozme were constructed in which several stabilizing disulfide bridges were combined in the same protein. The figure shows a schematic view of the a-carbon of T4 lysozyme and the locations of the disulfide bonds introduced. The inset shows the sizes of the loops formed by the respective disulfide bonds. (b) Differences in melting temperatures (ATm) of single-, double- and triple-disulfide-bonded lysozymes relative to wildtype lysozyme. The solid bars show the observed ATmsof the oxidized and reduced forms of the mutant lysozymes. The broken bars for the multiply bridged proteins correspond to the sums of the ATmSfor the constituent singly bridged proteins, showing additivity of mutational effects. These data demonstrate that a combination of stabilizing disulfide bonds can dramatically enhance protein stability. Reproduced from [52] and [55] with permission.

tion to what would be expected from the usual saltingout and salting-in effects on common globular proteins. Halophilic proteins show a specific requirement for high salt concentration (even saturated KC1) and they undergo denaturation if the solvent salt concentration falls below a certain value (e.g. 2.5 M KCl). According to the saltingout effect of apolar residues, halophilic proteins appear to be less hydrophobic than their common protein counterparts, and they possess polar, strongly hydrated amino acid residues at the protein surface [61]. Zaccai and Eisenberg [62 °] have studied in detail the stabilization mechanism of halophilic malate dehydrogenase, concluding that the hydrophobicity of the protein core is too weak to stabilize the folded structure and that in molar KCl the protein forms an exceptionally stable complex with hydrated salt ions. In molar KPO4, however, the stabilization model is predominantly of the salting-out type. Numerous cellular processes are controlled by precise levels of Ca2 + concentrations, as these ions are bound by a variety of intra- and extracellular proteins of different structure and biological role. One of the reasons why nature has chosen Ca2+ as regulator of protein function and stability may be the variability of its coordination. Whereas in organic complexes Ca2+ binds up to nine ligands, with a preference for seven or eight, in proteins the packing of residues around the cation re-

stricts the number of possible ligands. This variability of coordination leads to quite varied binding algmities (103_108 M - 1). In a recent study [63°], the detailed stereochemistry of the tightly bound Ca 2 + of Aspergillus niger ~z-amylase has been determined by X-ray crystallography and it was shown that eight protein ligands are involved in the ion binding. A compilation and discussion of the varied stereochemistries of calcium-binding sites in globular proteins as determined by X-ray crystallography are presented [63°]. Secondary structural features of amino acid residues in proteins providing ligands to metal ions (Ca, Zn) have also been analyzed [64°]. The stabilizing role of protein-bound Ca2+ has been documented extensively with a number of proteins (e.g. cz-lactalbumin, staphylococcal nuclease, parvalbumin III from carp muscle, a-amylase, subtilisin proteases, trypsin, concanavalin A, troponin, calmodulin and calbindin). Thermolysin, the highly stable neutral protease produced by Bacillus thermoproteolyticus, binds more Ca2 + than the thermolabile, mesophilic proteases [65]. It has been demonstrated that, among the four calcium ions bound to thermolysin, Ca-4 (bound at Asp200) is the most weakly bound ion and its role is to protect the corresponding ion-binding loop from autolytic inactivation [66]. Recently, the role of calcium in the stabilization of

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Proteinengineering thermitase, an extracellular protease isolated from Thermoactinomycetes vulgaris, has been studied by both model-building experiments [67] and X-ray crystallography [68]. Thermitase is the most thermostable member of the subtilisin family, which includes subtilisin Carlsberg, subtilisin Novo and proteinase K. The molecular details of the structure of thermitase, modelled on the basis of sequence homology with subtilisin Novo, were used to predict possible mechanisms of protein thermostability. A proposal was made that the thermitase molecule is not stabilized by salt bridges and/or hydrophobic interactions, but that an unusually tight binding of Ca2 + near Asp57 is most likely to be the major cause of thermostability [67]. Interestingly, this proposal has been substantiated by the experimentally determined high-resolution structure of thermitase [68]. In analogy to the therrnolysin case (see above), it may be that calcium binding in thermitase contributes to the protein stability by reducing the flexibility of the protein and its susceptibility to local and/or partial unfolding, followed by autolysis. The achievement of protein stabilization by the incorporation of ion-binding sites into proteins through genetic engineering is an attractive possibility, as the bridging function exerted by bound ions within a globular protein correlates with that of disulfide bonds (see above). An improved Ca2 +-binding site has been engineered into subtilisin by introducing negatively charged side chains in the vicinity of a weakly bound Ca2 + through the replacement of Pro172 and Gly131 by aspartic acid residues [69]. These changes were found to increase both the alTmity of the Ca2 +-binding site and the thermal kinetic stability. Interestingly, these effects were roughly additive. A Ca2 +-binding site has also been created in human lysozyme by the replacement of both Gln86 and Ala92 with aspartic acid residues by site-directed mutagenesis [70]. This lead to a mutant lysozyme which was more stable to heat and proteolytic digestion. Analogous stabilization effects were achieved in B. subtilis neutral protease in which the surface loop extending from amino acid residue 188 to residue 194 had been replaced by the 10-residue segment which, in the homologous polypeptide chain of thermolysin, binds Ca-4 (see above) [71"]. Engineering of metal-ion-binding sites into proteins is dealt with more thoroughly by Tainer et al. (this issue, pp 582-591).

Stability of thermophilic proteins Numerous studies carried out in the past 2 decades on the functional and molecular properties of thermophilic enzymes have established dearly that enzymes isolated from thermophilic microorganisms (growing optimally at 60-100°C) are generally much more resistant to heat and most common protein denaturants than their counterparts from mesophilic sources [72-75,76..]. All of the current evidence indicates that the enhanced stability of thermophilic enzymes cannot be attributed to a common determinant, but is the result of a variety of stabiliz-

ing effects including hydrophobic interactions, ionic and hydrogen bonding, disulfide bonds and metal binding. Similar forces and interactions also stabilize mesophilic proteins [67,77,78,79°]. On the basis of a detailed comparative study of sequences and structures of several thermophilic and mesophilic enzyme molecules, it has been proposed that a decreased flexibility and increased hydrophobicity in s-helical regions are the main stabilizing factors [78]. Frequent amino acid exchanges in thermophilic proteins tend to increase the alanine content of helices. Other favorable replacements include serine to alanine and lysine to arginine in thermophilic lactate dehydrogenase, glycine to alanine and serine to alanine in glyceraldhyde-3-phosphate dehydrogenase, and valine to alanine and lysine to alanine in triosephosphate isomerase [77,78]. Thus, it is interesting to observe the quite common exchanges of Xaa with alanine residues in helical segments of thermophilic enzymes [78]. Genetic techniques involving this particular amino acid replacement have been used to engineer protein stabilization (see above). A number of studies have demonstrated that genes from thermophiles can be expressed in mesophiles such as Escherichia coli and, using standard cultivation techniques, desired thermostable enzymes produced (see [79"] for references). Moreover, a method has been described for rapidly generating thermostable enzyme variants by introducing the gene coding for a given mesophilic enzyme into a thermophile (B. stearothermophilus) and then selecting variants retaining the enzymatic activity at the higher growth temperature of the thermophile. Using these procedures, thermostable variants of some proteins have been produced successfully [80,81]. Because the metabolic activities of thermophilic bacteria are similar to those of mesophiles, any enzyme already found in a mesophilic bacterium would also be expected to be found in a thermophilic one. Thus, considering the wide variety of bacteria living in extreme environments at about 100°C or above [82], there appears to be an unlimited variety of stable enzymes that can be isolated and exploited successfully in biotechnology. One of the most important and successful practical applications of thermophilic enzymes involved the use of DNA polymerase from Therm~ aquaticus for the enzymatic amplification of I)NA fragments (polymerase chain reaction) [83]. Moreover, thermophilic enzymes have a number of useful applications for basic research, as they are most suitable protein models for addressing a number of relevant problems in protein stability and folding.

Conclusion This brief summary of selected issues and papers from the past year documents the advances achieved in devising successful strategies for stabilizing proteins. Numerous site-directed mutagenesis experiments have allowed the role and magnitude of the stabilizing interactions leading to protein structure and stability to be dis-

Analysis and modulation of protein stability Fontana sected. The cumulative effect of mutations in protein stability has been demonstrated convincingly, thus indicating the potential for designing and producing genetically new proteins and enzymes of desired stability. Nevertheless, because a general and quantitative comprehension of the protein stability problem is still lacking, this area will continue to be the subject of very intensive investigation using a variety of physicochemical and biological techniques and approaches. The outcomes of such studies should be rewarding from both fundamental and practical points of view.

Acknowledgements I am most grateful to B Matthews, N Pace, R Wetzel, K Dill and R Saner for sending me reprints and preprints of their studies. The excellent assistance of Mrs A Mocavero in the preparation of the manuscript is also gratefully acknowledged. This work was supported by a grant of the Special Project of Italian CNR on Biotechnology and Bioinstmmentation.

dividual protein stabilizing interactions are discussed, concluding that hydrogen bonding is a major contribution to protein stability. Recent experimental work appears to demonstrate that hydrophohic interactions are not the dominant stabilizing forces. 11. MATII-IEWSBW: Mutational Analysis of Protein Stability. Curr •. Opin Struct Biol 1991, 1:17-21. An up-to:date review describing the most recent results of protein engineering studies aimed at understanding the problem of protein folding and stability, Changes in protein stability caused by mutations can often be rationalized, making it possible to design and produce genetically new proteins of desired stability.

12.

SCHELLMANJA: The Thermodynamic Stability of Proteins. A n n u Rev Biophys Chem 1987, 16:115-137.

13.

BECKTELWJ, SCHELLMANJka Protein Stability Curves. Biopol3* mers 1987, 26:1859-1877.

14. DILL K& Dominant Forces in Protein Folding. Biochemistry •• 1990, 29:713/',7155. An up-to-date and excellent discussion of the forces which dictate protein structure. It is concluded that proteins, in general, should be tolerant to amino acid substitutions, as a given native structure should be encodable by many different sequences. 15.

PACE CN, GRIMSLEY GPC Ribonuclease T1 is Stabilized by Cation and Anion Binding. Biochemistry 1988, 27:3242-3246.

16. •

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1. CREIGHTONTE: Protein Folding. Biochem J 1990, 270:1-16. • A review describing recent results which highlight the actual folding process of small, single-domain proteins. 2. JAENICKEPc Protein Folding: Local Structures, Domains, Sub• units and Assemblies. Biochemistry 1991, 30:3147-3161. A thorough and up-to-date review of our current understanding of the mechanism of protein folding. The role of chaperones in promoting protein folding is also discussed. 3.

DE GRADOWF: Design of Peptides and Proteins. Adv Protein Chem 1988, 39:51-124.

4.

MATrHEWSBW: Genetic and Structural Analysis of the Protein Stability Problem. Biochemistry 1987, 26:6885-6888.

5.

SHORTLED: Probing the Determinants of Protein Folding and Stability with Amino Acid Substitutions. J Biol Chem

6.

AIBERT: Mutational Effects on Protein Stability. A n n u Rev Biochem 1989, 58:765-798.

7.

ALBERT: Stabilization Energies of Protein Conformation. in

PACE CN, LAURENTSDV, THOMSON'J& pH Dependence of the Urea and Guanidine Hydrochloride Denaturation of Ribonuclease A and Ribonuclease T1. Biochemistry 1990, 29:2564-2572. The solvem denaturation of ribonucleases A and T1 has been investigated. The conformations assumed by the unfolded protein depends on its amino acid sequence. The role of the unfolded state in the thermodynamic stability of globular protein is discussed. 17.

18. ~he

PACE CN: Conformational Stability of Globular Proteins. rends Biochem Sci 1990, 15:14-17. methods currently employed to analyze protein stability are reMewed using ribonuclease T1 as a test case. Factors that contribute to protein stability are discussed brietly. 19.

PACECN: Measuring and Increasing Protein Stability. Trends Biotechnol 1990, 8:93-98. A review describing recent developments in the methods used for measuring the conformational stability of globular proteins. Approaches for enhancing protein stability are also discussed. 20.

1988, 264:5315-5318.

Prediction of Protein Structure a n d the Principles of Pro tein Conformation edited by Fasman GD [book]. New York: Plenum Press 1989, pp 161-192. 8.

PAKULA AA~ SAUERRT: Genetic Analysis of Protein Stability and Function. Annu Rev Genet 1989, 23:289-310.

9. NOSOHY, SEKIGUCHIT: Protein Engineering for Thermosta• bility. Trends Biotechnol 1990, 8;16-20. Describes the use of protein engineering for both exploring the molecular basis of protein stability and producing genetically more stable proteins. 10. CREIGHTONTE: Stability of Folded Conformations. Curr Opin •. Struct Biol 1991, 1:5-16. An excellent review describing recent developments towards an understanding of the physical basis of the stability of globular proteins, in-

FREIREE, VAN OSDOL "WRY, MAYORGA OL, SANCHEZ-RUIZJM: Calorimetrically Determined Dynamics of Complex Unfolding Transitions in Proteins. Annu Rev Biophys Biophys Chem 1990, 19:159-188.

WETZELR, PERRyJ, MULKERRINMG, RANDALLM: Unfolding and Inactivation: Genetic and Chemical Approaches to the Stabilization of T4 Lysozyme and Interferon-Gamma Against Irreversible Thermal Denaturation. In Protein Design a n d the Development of New Therapeutics a n d Vaccines edited by Poste G, Crooke S [book]. New York: Plenum Press 1989, pp 79-115.

21. ••

BELL JA, DAO-PIN S, FABER R, JACOBSON R, KARPUSAS M, MATSUMURAM, NICHOLSONH, PJURAPE, TRONRUD DE, WEAVER LH ET AL.: Approaches Toward the Design of Proteins of Enhanced Stability. in The Use of Crystallography in the D e sign of Antiviral Agents, edited by Laver WG, Air GM [book]. Orlando, Florida: Academic Press 1991, in press. Reviews the studies on a great variety of mutants ofT4 lysozyme. On the basis of the results obtained using this model protein, general strategies for enhancing protein stability are proposed and discussed. 22. •

ALEXANDRESCUAT, HINCKAP, MARKLETJL: Coupling Between Local Structure and Global Stability of a Protein: Mutants of Staphylococcal Nuclease, Biochemistry 1990, 29:4516-4525. Seven mutants of nuclease were employed to study the equilibrium between two different substrates of native as well as unfolded enzyme. 23. •

SONDEJ, SHORTLED: Accommodation of Single Amino Acid Insertions by the Native State of Staphylococcal Nuclease. Proteins 1990, 7:299-305.

557

558

Protein engineering Single alanine and glycine insertions were introduced into nuclease. Generally, it was found that the enzyme is able to accommodate the extra residue without difficulty. Amino acid insertion appears to be a novel method for engineering proteins. SHORTLED, STITES WE, MEEKER AK: Contributions of the Large Hydrophobic Amino Acids to t h e Stability of Staphylococcal Nuclease. Biochemistry 1990, 29:8033-8041. A collection of 83 mutants of nuclease were constructed by substituthag hydrophobic residues with alanine or giycine and their stability to guanidine hydrochloride-mediated unfolding was investigated. The results obtained indicate that substitutions which destabilize the protein have an effect on the structure and free energy of the denatured state.

34.

NICHOLSONH, BECKTELWJ, MATla-IEWSBW: Enhanced Protein Thermostability from Designed Mutations that Interact w i t h c¢-Helix. Nature 1988, 366:651~556.

35.

ALBERT, BEL JA, SUN DP, NICHOLSON H, WOZNIAKJA, COOK SP, MATrHEWS BW: Replacements of Pro 86 in Phage T4 Lysozyme Extend an c~-Helix b u t do n o t Alter Protein Stability. Science 1988, 239:631~535.

24. •

25. ..

SAUERRT, JORDAN SR, PABO CO: Lambda Repressor: a Model System for Understanding Protcin-DNA Interactions and Protein Stability. Adv Protein Chem 1990, 40:1~51. This review presents an excellent summary of the results of protein engineering studies of £-repressor, and an analysis of the residue changes that increase the thermostability of the protein. The significance of the results obtained for an understanding of protein structure-function stability are discussed. 26.

MCNUTTM, MULLINSLS, RAUSHELFM, PACE CN: Contribution of Histidine Residues to t h e Conformational Stability of Ribonuclease T1 and Mutant Glu58 --+Ala. Biochemistry 1990, 29:7572-7576.

27. •

DECLERKN, JOYET P, GAILLARDINC, MASSONJM: Use of Amber Suppressors to Investigate t h e Thermostability of Bacillus licheniformis Alpha-Amylase. Amino Acid Replacements at 6 Histidine Residues Reveal a Critical Position at His133. J Biol Chem 1990, 265:15481-15488. The results of this paper s h o w that the thermostability of a highly thermostable enzyme is not maximized, but can be enhanced using laboratory experiments. Thus, it seems that it is advantageous for living organisms to have proteins for which the folded, biologically active conformation is marginally more stable than the unfolded, inactive conformation. 28.

HOLML, KOlVULAAK, LEHTOVAARAPM, HEMMINKIA, KNOWLES • JKC: Random Mutagenesis Used to Probe t h e Structure and Function of Bacillus stearothermophilus Alpha-Amylase. Protein Eng 1990, 3:181-189. An efficient random mutagenesis method was used to produce 98 mutants of the c~-amylase and the corresponding effects on the functional properties were investigated. Exposed loops are found to be fairly tolerant to amino acid substitutions. 29.

MATTHEWSBW, NICHOLSONH, BECKTELWJ: Enhanced Protein Thermostability from Site-Directed Mutations that Decrease the Entropy of Unfolding. Proc Natl Acad Sci USA 1987, 84:6663-6667.

30.

IMANAKAT, SHIBAKAKIM, TAKAGI M: A N e w Way of Enhancing the Thermostability o f Proteases. Nature 1986, 324:695-697.

31.

HECHTMH, STURTEVANTJM, SAUER RT: Stabilization of £-Repressor Against T h e r m a l Denaturation by Site-Directed Gly --+Ala Changes in 0t-Helix 3. Proteins 1986, 1:43-46.

32. •

GANTERC, PLOCKTHUNA: Glycine to Alanine Substitutions in Helices of Glyceraldehyde-3-Phosphate Dehydrogenase: Effects o n Stability. Biochemistry 1990, 29:9395-9402. The protein stabilization effects of glycine-to-alanine replacements in helical regions of the oligomeric enzyme, glyceraldehyde-3-phosphate dehydrogenase, are determined. One mutation (Gly166Ala) stabilizes the protein, whereas the others are neutral or destabilizing. It is concluded that the glycine-to-alanine substitution does not stabilize the enzyme by entropic effects, but rather by filling an internal cavity in the native enzyme. 33. •

DAO-PINS, BAASEWA, MATTHEWSBW: A Mutant T4 Lysozyme (Vall31~Ala) Designed to Increase Thermostability by t h e Reduction of Strain w i t h i n an Alpha-Helix. Proteins 1990, 7:198-204. The valine-to-alanine substitution in T4 lysozyme produces a modest gain in protein stability. The reduction in helix strain is partially offset by the loss of hydrophobic interactions and by entropic effects.

36. •

PADMANABHANS, MARQUSEES, RIDGEWAYT, LAUE TM, BALDWIN RL: Relative Helix-Forming T e n d e n c i e s of Nonpolar Amino Acids. Nature 1990, 344:268-270. The results of this paper show that the helix-forming tendency of a particular amino acid depends on the sequence context in which it occurs. The restriction of side-chain rotamer conformation in the a-helix is an important factor in determining the helix propensity. 37. .

CHAKRABARTIYA, SCHELLMANJ ~ BALDWIN RL: Large Differe n c e s in t h e Helix Propensities of Alanine and Glycine. Nature 1991, 351:586-588. Substitution experiments with a 17-residue model helical peptide reveal that the ratio of the helix propensities of alanine to glycine is ap proximately 100. The helix-destabilizing effect of an alanine-to-glycine substitution depends largely on its position in the helix. 38.

MERUTKAG, SHALONGOW, STELLWAGENE: A Model Peptide with Enhanced Helicity. Biochemistry 1991, 30:4245-4248. ~ h e effect of amino acid replacements on the helix-coil equilibrium of a small peptide is studied. Evidence is provided that a synthetic model peptide can be engineered to be nearly completely helical in aqueous solution. 39. ••

ZHANGX-J, BAASE WA, MAT17-mWSBW: Toward a Simplification o f t h e Protein Folding Problem: a Stabilizing Polyalanine a-Helix Engineered in T4 Lysozyme. Biochemistry 1991, 30:2012-2017. A series of alanines introduced into T4 lysozyme leads to protein stabilization. This study shows that polyalanination might provide a means of simplifying the protein-folding problem. 40. ..

BOWIEJU, REIDHAAR-OLSONJF, LIM WA~ SAUERRT: Deciphering t h e Message in Protein Sequences: Tolerance to Amino Acid Substitutions. Science 1990, 247:1306-1310. Random mutagenesis is carried out in order to generate a list of tolerated amino acid substitutions in a helix of ~v-repressor. The technique is useful for identifying the residues that are buried or surface-exposed in a protein structure. Tolerance to exchange is high for exposed residues. 41. ..

REIDHAAR-OISONJF, SAUER RT: Functionally Acceptable Substitutions in T w o c~-Helical Regions of ~-Repressor. Proteins 1990, 7:306-316. The pattern of allowed amino acid exchanges at each position in two helices of ~-repressor permits the importance of single residues in the function and stability of the protein to be assessed. Overall, the data show the degeneracy of the folding code contained in the amino acid sequence of a protein. 42.

LIM W ~ SAUER RT: Alternative Packing Arrangements in t h e Hydrophobic Core of Lambda Repressor. Nature 1989, 339:31-36.

43.

BASHFORDD, CHOTHIAC, LESKAM: Determinants of a Protein Fold: Unique Features of t h e Globin Amino Acid Sequences. J Mol Biol 1987, 196:199-216.

44.

BORDOD, ARGOS P: Evolution of Protein Cores. Constraints in Point Mutations as Observed in Globin Tertiary Structures. J Mol Biol 1990, 211:975-988.

45. •

BOWIEJU, CLARKE ND, PABO CO, SAUER RT: Identification of Protein Folds: Matching Hydrophobicity Patterns of Seq u e n c e Sets w i t h Solvent Accessibility Patterns of K n o w n Structures. Proteins 1990, 7:25%264. The pattern of hydrophobicity values derived from a set of related prorein sequences correlates with the linear pattern of side-chain solvent accessibility values. The information from aligned sequences can be used to identify the tertiary fold adopted by a set of protein sequences. 46.

AKKE M, FORSEN S: Protein Stability and Electrostatic Interactions B e t w e e n Solvent-Exposed Charged Side Chains. Proteins 1990, 8:23-29.

Analysis and modulation of protein stability Fontana 47. ,.

ANDERSONDE, BECKTELWJ, DAHLQUISTFX~7: pH-Induced Denaturation o f Proteins: A Single Salt Bridge Contributes 3-5 Kcal/Mol to the Free Energy o f Folding of T4 Lysozyme. Biochemistry 1990, 29:2403-2408. The energetics of a salt bridge (Asp70-His31) in T4 lysoz3ane have been examined by site-directed mutagenesis and nuclear magnetic resonance techniques. It is shown that the electrostatic contributions of ionizable groups to the stabilization of the folded state of the protein can be assessed directly by measuring apparent pKa values of ionizing groups. SERRANOL, HOROVITZ A, AVRON B, BYCROFT M, FERSHT A_R: Estimating t h e Contribution of Engineered Surface Electrostatic Interactions to Protein Stability b y Using DoubleMutant Cycles. Biochemistry 1990, 29:9343-9352. A method is presented for estimating the coulombic interaction energy between two charged residues by using a double-mutant cycle. Electrostatic effects are estimated from changes in protein unfolding energies.

A computer-simulated neural network is used for predicting whether or not a cysteine participates in a disulfide bond. The method is useful for designing the introduction of disulfide bridges into proteins. 59.

ARAKAWAT, TIMASHEFF SN: Preferential Interactions of Proteins w i t h Salts in C o n c e n t r a t e d Solutions. Biochemistry 1982, 21:6545-6552.

60.

VON HIPPEL PH, SCHLEICH T: The Effects of Neutral Salts o n t h e Structure and Conformational Stability of Macromolecules in Solution. In Structure and StabiliO~ of Biological Macromolecules edited by Timasheff SN, Fasman GD [hook]. New York: Marcel Dekker 1969, pp 417-574.

61.

EISENBERGH, WACHTEL EJ: Structural Studies of Halophilic Proteins, Ribosomes and Organelles of Bacteria Adapted to Extreme Salt Concentrations. Annu Rev Biophys Biopbys Chem 1987, 16:69--92.

48. •

49. WELLSJ& Additivity of Mutational Effects in Proteins. BiD •• chemistry 1990, 29:8509-8517. An excellent and up-to-date review of data demonstrating that singlepoint mutations of proteins show additivity effects in protein function and stability. 50.

AHERNTJ, KLmANOVAM: T h e M e c h a n i s m o f Irreversible Enzyme Inactivation at 100°C. Science 1985, 228:1280-1284.

51.

WETZELR: Harnessing Disulfide Bonds Using Protein Engineering. Trends Biochem Sci 1987, 12:478-482.

52.

MATSUMURAM, MATrHEWS BW: Stabilization of Functional Proteins by t h e Introduction of Multiple Disulphide Bonds. In Methods in Enzymology, Molecular Design and Modeling edited by Langone JJ [hook]. Orlando, Florida: Academic Press 1991, in press.

53.

PERRYLJ, WETZEt R: Disulphide Bond Engineered into T4 Lysozyme: Stabilization of t h e Protein Toward T h e r m a l Inactivation. Nature 1984, 226:555-557.

54. •

PIURAPE, MATSUMURAM, WOZNIAKJA, MATrHEWS BW: Structure of a Thermostable Disulfide-Bridge Mutant o f Phage T4 Lysozyme Shows that an Engineered Cross-Link in a Flexible Region does n o t Increase t h e Rigidity o f t h e Folded Protein. Biochemistry 1990, 29:2592-2598. The crosslinked T4 lysozyme has overall crystallographic temperature factors very similar to those of the wild-type enzyme, indicating that the introduction of the disulfide b o n d does not impose overall rigidity to the folded ptTotein. It is concluded that disulfide bridges increase the stability of proteins by reducing the conformational entropy of the unfolded state. 55.

MATSUMURA M, SIGNORG, MAaTHEWSBW: Substantial Increase of Protein Stability by Multiple Disulphide Bonds. Nature 1989, 342:291-293.

NISHIKAWAS, ADIWINATAJ, MORIOKAH, FUJIMURA.T, TANAKAT, UESUGIS, HAKOSHIMAT, TOM1TAK, NAKAGAWAS, IKEHARAM: A Thermoresistant Mutant of Pdbonuclease T1 having T h r e e Disulfide Bonds. Protein Eng 1990, 3:443-448. A more stable and active mutant of ribonuclease T1 containing a third disulfide bond between residues 24 and 84 is described. However, the mutant is less stable than the wild-type toward irreversible denaturation at 100°C as a result of interchange of disulfide bonds.

62. •

ZACCAIG, EISENBERG H: Halophilic Proteins and t h e influe n c e of Solvent o n Protein Stabilization. Trends Biochem Sci 1990, 15:333-337. Discusses the mechanism by which halophilic proteins have adapted their interactions with solvents in order to be both folded and enzymatically active under their normal conditions of nearly saturated KC1. 63. •

BOEL E, BRADY L, BRZOZOWSKI AM, DEREWENDAZ, DODSON GG, JENSEN VJ, PETERSEN SB, SWIFT H, THIM L, WOLDIKE HF: Calcium Binding in m-Amylase: An X-Ray Diffraction Study at 2.1-J~-Resolution of T w o Enzymes from Aspergillus. BiD chemistry 1990, 29:6244-45249. The detailed stereochemistry of the tighdy b o u n d Ca2 + of Aspergillus niger or-amylase has been determined by X-my crystallogmphTf. It is shown that eight protein ligands are involved in the ion binding. The stereochemistry of Ca 2 +-binding sites in proteins is also reviewed. The data presented could be a useful guide for designing Ca2 + -binding sites by protein engineering experiments. 64. •

CHAKRABARTIP: Interaction of Metal Ions w i t h Carboxylic and Carboxamide Groups in Protein Structures. Protein Eng 1990, 4:49-56. An analysis of the geometry of metal binding of aspartic acid and glutamic acid side chains in proteins. Calcium ions can be b o u n d at the end of helices. The results of this study will be useful for designing suitable ligands for metal ions in proteins. 65.

ROCHE RS, VOORDOUW G: T h e Structural and Functional Roles of Metal Ions in Thermolysin. CRC Crit Rev Biochem 1978, 5:1 23.

66.

FASSlNA G, VITA C, DALZOPPO D, ZAMAI M, ZAMBONIN M, FONTANA A: Autolysis of Thermolysin: Isolation and Characterization of a Folded Three-Fragment Complex. Eur J Biochem 1986, 156:221-228.

67.

FROMMELC, SANDER C: Thermitase, a Thermostable Subtilisin: Comparison of Predicted and Experimental Structures and t h e Molecular Cause o f Thermostability. Proteins 1989, 5:22-37.

68.

BETZEL C, TEPLYAKOV AM, HARUTYUNYANEH, SAENGER W, WILSON KS: Thermitase and Proteinase K: a Comparison of t h e Refined Three-Dimensional Structures of t h e Native Enzymes. Protein Eng 1990, 3:161-172.

69.

PANTOLIANOMW, WHITLOWM, WOOD JF, ROLLENCEML, FINZEL BC, GILIaLANDGL, POULOS TL, BRYANPN: The Engineering of Binding AtFnity at Metal Ion Binding Sites for t h e Stabilization of Proteins: Subtilisin as a Test Case. Biochemistry 1988, 27:8311-8317.

70.

KUROFaR, TANIYAMAY, SEKO C, NAKAMURAH, KIKUCHI M, IKEHARA M: Design and Creation of a CaX+-Binding Site in H u m a n Lysozyme to Enhance Structural Stability. Proc Natl Acad Sci USA 1989, 86:6903-6907.

71. •

TOMA S, CAMPAGNOLI S, MARGARIT I, GIANNA R, GRANDI G, BOLOGNESIM, DE FILIPPISV, FONTANAA: Grafting of a CalciumBinding Loop of Thermolysin to Bacillus subtilis Neutral Protease. Biochemistry 1991, 30:97-106.

56. •

57.

TAKAG1H, TAKAHASHIT, MOMOSE H, INOUYE M, MAEDA Y, MATSUZAWA H, OHTA T: E n h a n c e m e n t of t h e Thermostability of Subtilisin E by Introduction of a Disulfide Bond Engineered on t h e Basis of Structural Comparison w i t h a Thermophilic Serine Protease. J Biol Chem 1990, 265:6874-6878. Disulfide bonds are introduced into subtilisin E on the basis of the structure of the stable, homologous protease from Thermus aquaticus. The disulfide-containing mutant shows enhanced stability and impaired catalytic ett]ciency. •

58. •

MUSKAL SM, HOLBROOK SR, KIM SH: Predictiion of t h e Disulfide-Bonding State of Cysteine in Proteins. Protein Eng 1990, 3:667-$572.

559

560

Protein engineering A seven-residue surface loop (~-loop) of the mesophilic protease was replaced by a 10-residue segment which, in the homologous thermolysin molecule, binds calcium. The mutant protease binds calcium and is stabilized against thermal denaturation. This illustrates the feasibility of engineering metal-ion-binding sites into proteins. 72.

ZtmERG (ED): Enzymes and Proteins from Thermophilic Microorganisms [book]. Basel: Birkfiuser 1976.

73.

JAENICKER: Enzymes Under Extremes of Physical Conditions. A n n u Rev Biopbys Bioeng 1981, 10:1~57.

74.

FONTANAA: Structure and Stability of Thermophilic Enzymes: Studies on Thermolysin. Biopbys Cbem 1988, 29:181-193.

75.

KRISTJANSSONJK: Thermophilic Organisms as Sources of Thermostable Enzymes. Trends Biotechnol 1989, 7:349-353.

76. JAEImCKER, ZAVODSKYP: Proteins Under Extreme Physical o• Conditions. FEBS Lett 1990, 268:344-349. Current views on the stabilization mechanisms of proteins derived from extremophilic microorganisms are reviewed. The low-temperature inactivatiort/unfolding of proteins is also discussed. 77.

ARGOS P, ROSSMANN MG, GRAU UM, ZUBER H, FRANK G, TRATSCHINJD: Thermal Stability and Protein Structure. Bio chemistry 1979, 18:5698-5703.

78.

MENI~NDEZ-ARIASL, ARGOS P: Engineering Protein Thermal Stability. Sequence Statistics Point to Residue Substitutions in 0~-Helices.J Mol Biol 1989, 206:397-406.

79. •

FONTANAA: HOW Nature Engineers Protein (Thermo) Stability. In Life Under Extreme Conditions: Biochemical Adaptation edited by di Prisco G [book]. Berlin-Heidelberg: Springer Verlag 1991, pp 89-113. A review of recent results from the analysis of correlations between structure and stability in thermophilic enzymes. Of interest, typical amino acid replacements (e.g. glycine to alanine) found in thermophilic enzymes are those that are used to engineer protein stability by sitedirected mutagenesis experiments. 80.

MATSUMURA M, AIBA S: Screening for Thermostable Mutant of Kanamycin Nucleotidyltransferase by the Use of a Transformation System for a Thermophile (B. stearothermophilus). J Biol Chem 1985, 260:15228-15233.

81.

LIAOH, MCKENZIET, HAGEMANR: Isolation of a Thermostable Enzyme Variant by Cloning and Selection in a Thermophile. Proc Natl A c a d Sci USA 1986, 83:576-580.

82.

BROCK TD: Life at High Temperature. 230:132-138.

83.

SAWdRK, GELFANDDH, STOFFELS, SCHARFSJ, HIGUCHIR, HORN GT, MULUSKB, EHRLICHHA: Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science 1988, 239:487-491.

Science

1985,

A Fontana, Department of Organic Chemistry and CRIBI Biotechnology Centre, University of Padua, Via Trieste 75, 35121 Padua, Italy.

Analysis and modulation of protein stability.

Numerous site-directed mutagenesis experiments have provided new insights into the stabilizing role of the individual forces and interactions within a...
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