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Two exposed amino acidresidues confer thermostability on a cold shock proteinDieter Perl1, Uwe Mueller2,3, Udo Heinemann2,3 and Franz X. Schmid1

1Biochemisches Laboratorium, Universität Bayreuth, D-95440 Bayreuth,Germany. 2Forschungsgruppe Kristallographie, Max-Delbrück-Centrumfür Molekulare Medizin, Robert-Roessle-Str. 10, D-13125 Berlin,Germany. 3Institut für Chemie – Kristallographie, Freie Universität Berlin,Takustr. 6, D-14195 Berlin, Germany.

Thermophilic organisms produce proteins of exceptionalstability. To understand protein thermostability at the mol-ecular level we studied a pair of cold shock proteins, one ofmesophilic and one of thermophilic origin, by systematicmutagenesis. Although the two proteins differ in sequenceat 12 positions, two surface-exposed residues are responsi-ble for the increase in stability of the thermophilic protein(by 15.8 kJ mol-1 at 70 °C). 11.5 kJ mol-1 originate from apredominantly electrostatic contribution of Arg 3 and5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at thecarboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing theGlu residues at positions 3 and 66 to Arg and Leu, respec-tively. The variation of surface residues may thus provide asimple and powerful approach for increasing the ther-mostability of a protein.

Proteins from thermophilic organisms are important toolsin biochemistry and provide conceptual insights for rationalprotein design. The molecular determinants of their high ther-mostability are, however, not well understood. This is becausemesophilic and thermophilic paralogs typically differ in

sequence at many positions1 and because irreversible unfold-ing often impairs quantitative thermodynamic analyses2 of thefactors that confer incremental increases in stability. Here weused the cold shock proteins3,4 from the mesophile Bacillussubtilis (Bs-CspB) and from the thermophile Bacillus caldolyti-cus (Bc-Csp) (Fig. 1) to investigate the role of each of theirvariant amino acids in the enhanced stability of the ther-mophilic protein. Bs-CspB and Bc-Csp are small, monomericproteins of 67 and 66 residues, respectively, without disulfidebonds, cofactors or tightly bound ligands. They differ insequence at 12 positions, all of which map to the protein sur-face. The crystal structures, solved at 2.45 Å (Bs-CspB)5 and1.17 Å (Bc-Csp)6 resolution, reveal that both have virtuallyidentical backbone conformations. Both proteins unfoldreversibly in a monomolecular native (N) unfolded (U)two-state reaction7 and are thus well suited for quantitativethermodynamic studies. The midpoint of the thermal unfold-ing transition (TM) of Bc-Csp is 76.9 °C, 23 degrees higher thanthe TM of Bs-CspB. Because of these properties and the smallnumber of sequence differences between the two proteins, Bc-Csp and Bs-CspB make an excellent pair of models foridentifying the origins of thermostability at the molecularlevel by a combination of systematic mutagenesis and thermo-dynamic measurements.

Molecular dissection of Bc-Csp thermostabilityWe produced 12 variants of the thermophilic protein Bc-Csp,each containing a single amino acid substitution at a positioncorresponding to one of the 12 sequence differences betweenthe two proteins. These were made to determine the individualcontributions of each of these amino acids to the increasedGibbs free energy of denaturation (∆GD) of Bc-Csp. Eleven of

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Fig. 1 The mesophilic cold shock protein Bs-CspB differs from its thermo-philic homolog, Bc-Csp, at 12 sequence positions. a, Cα trace derivedfrom the crystal structure of Bc-Csp6 in two orientations with side chainsthat are different in Bs-Csp added (green). The backbone conformationof Bs-CspB5 is nearly identical. b, Sequence alignment of the cold shockproteins. In Bc-Csp the C-terminus is shortened by one residue, and 11amino acids are changed (green) with respect to the Bs-CspB sequence.Five of these changes involve charged residues.

Fig. 2 Thermal stability curves. a, Unfolding transitions of wild type Bc-Csp and three destabilized variants. b, Unfolding transitions of wildtype Bs-CspB and three stabilized variants in 100 mM Na cacodylate/HCl,pH 7.0, at protein concentrations of 4 µM. The fractions of native proteinobtained after a two-state analysis of the data are shown as a function oftemperature. The continuous lines show the results of the analysis.

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the variants contained single amino acid substitu-tions, and one was extended by an Ala 67, as in Bs-CspB (Fig. 1b). The thermal unfolding transi-tions of these proteins were followed by thedecrease in circular dichroism (CD). All transitionswere reversible, monophasic and independent ofprotein concentration, as expected for simpleN U two-state reactions; representative transi-tions are shown in Fig. 2a. Values for TM and ∆GD

were derived from the two-state analysis8 of thesethermal unfolding curves (Table 1). The ∆GD valuesare compared at 70 °C, which is near the optimalgrowth temperature of B. caldolyticus and 7 °Cbelow the TM of Bc-Csp. Moreover, this tempera-ture is in the thermal transition region of most ofthe variants. Here extrapolation can be avoidedand the values for ∆GD can be obtained with highaccuracy.

The differences in stability ∆∆GD between wildtype Bc-Csp and the individual variants are shownin Fig. 3. They reveal that the increased thermostability of Bc-Csp is not the sum of many small increments. Rather, itoriginates largely from the contributions of Leu 66 and, in par-ticular, Arg 3 (Fig. 2a), which alone accounts for ∼ 70% of theentire difference in thermodynamic stability between themesophilic and thermophilic proteins. Positions 3 and 66 areGlu residues in Bs-CspB. When the two mutations were combined (in the R3E/L66E double mutant of Bc-Csp) the TM

dropped by 32.3 °C and ∆GD by 20.7 kJ mol-1 (Fig. 2a, Table 1).Bc-Csp R3E/L66E is 4 kJ mol-1 less stable than expected fromthe sum of the individual mutations and thus even less stablethan the mesophilic protein Bs-CspB (Fig. 3). This additionaldestabilization probably results from the repulsion of the threeGlu residues at positions 3, 46 and 66. Such a cluster of nega-

tive charges is not present in either parent protein.Electrostatic repulsion should be screened by counter ions,and, indeed, in the presence of 2 M NaCl the destabilizationcaused by the R3E/L66E double mutation was reduced to -9.6 kJ mol-1, which is very close to the sum of the destabiliza-tions measured for the two individual variants R3E and L66Eat 2 M NaCl (-4.2 and -5.3 kJ mol-1, respectively; Table 1).

All other differences in sequence between the two proteinslead to changes in stability that are smaller than ±1.5 kJ mol-1.The only exception is Gln 2. Mutation to a Leu (as in themesophilic protein) actually stabilized the thermophilic pro-tein by 2.3 kJ mol-1 (Table 1).

The ∆∆GD values for all the individual mutations add up to -15.1 kJ mol-1, which is surprisingly close to the overall ∆∆GD

value between the mesophilic and the thermophilicparent proteins (-15.8 kJ mol-1). This additivity wasobserved not only at 70 °C (Table 1), but also at 50and 60 °C (data not shown).

Thermostabilization of Bs-CspB by twosurface mutationsTo test whether the mesophilic protein could be stabilized by the reciprocal mutations at positions 3and 66 we introduced Arg 3 and Leu 66 separately andin combination into Bs-CspB. The E3R mutation alonestabilized Bs-CspB by 11.1 kJ mol-1 (Fig. 2b, Table 1)and thus doubled the Gibbs free energy of stabilizationof Bs-CspB at 25 °C (11.3 kJ mol-1; ref. 7). The E3Rmutation stabilized Bs-CspB to the same extent as thereverse R3E mutation destabilized Bc-Csp (-11.5 kJ mol-1; Table 1). This is remarkable because

Fig. 3 Effect of all individual sequence differences on the stability of Bc-Csp. The first 12entries display the relative differences in stability (∆∆GD) caused by the individual substi-tution of the respective residues of Bs-CspB into Bc-Csp. The two bars at the right showthe destabilization of Bc-Csp and the stabilization of Bs-CspB, respectively, by the recipro-cal double mutations at positions 3 and 66. The relative stabilities of the parent proteinsBc-Csp and Bs-CspB are indicated by the horizontal lines.

Fig. 4 The residues that contribute most to the difference inthermal stability between Bs-CspB and Bc-Csp are located neareach other at the protein surface. In the stereo diagrams theprotein backbone and the side chain carbons are shown in darkand light gray, respectively. Side chain nitrogens are coloredblue, oxygens red and sulfurs yellow. Residues at positions 3, 46and 66, which are important for protein stability, have greencarbon atoms. a, Surface portion of the Bc-Csp structure6. Theorientation of the side chains of Arg 3 and Glu 46 in a secondprotein molecule present in the crystal structure (pink) permitsformation of a salt bridge. b, Surface portion of the Bs-CspBstructure5. The side chains of Glu 3 and Glu 66 are disordered inthe crystal structure and were modeled in low-energy confor-mations.

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these substitutions involve charge reversals, and Bs-CspB andBc-Csp differ in charge at six positions. The E66L mutationalso strongly stabilizes Bs-CspB (∆∆GD = 8.8 kJ mol-1; Table 1).In this case, the stabilization is even stronger than the corre-sponding destabilization of Bc-Csp by the L66E mutation(∆∆GD = -5.2 kJ mol-1). This additional stabilization of themesophilic protein occurs because L66E (as E3R) removes anunfavorable electrostatic repulsion between Glu 3 and Glu 66,which is present in wild type Bs-CspB. When both Arg 3 andLeu 66 were introduced into Bs-CspB the transition midpointincreased by 20.6 °C (Fig. 2b) and the stability increased by14.2 kJ mol-1. Thus, this double mutant of the mesophilic protein Bs-CspB is only 1.6 kJ mol-1 less stable than the ther-mophilic protein Bc-Csp (Fig. 3, Table 1).

Dominant electrostatic contribution of Arg 3The additional stability of Bc-Csp relative to Bs-CspB is largelyof electrostatic origin6; the difference in ∆∆GD between themdecreased by 7.4 kJ mol-1 (from 15.8 to 8.4 kJ mol-1) when theNaCl concentration was increased from 0 to 2 M (Table 1). Thedifference in ∆GD between wild type Bc-Csp and the R3E singlemutant also decreased by 7.3 kJ mol-1 (from 11.5 to 4.2 kJ mol-1)in 2 M NaCl, suggesting that the strong electrostatic contribu-tion to ∆∆GD is fully accounted for by the Arg to Glu difference.

In the high resolution crystal structure of Bc-Csp the asym-metric unit contains two protein molecules with alternativeconformations for the side chain of Arg 3. It is close to Glu 46(2.7 Å) in one molecule, but further apart (4.6 Å) in the other(Fig. 4). Thus, the extra electrostatic stabilization of the ther-

mophilic protein might not originate fromthe formation of an Arg 3–Glu 46 ion pair.This is borne out by the stability measure-ments of the mutants. In Bc-Csp a putativeArg 3–Glu 46 ion pair should be disruptedby the E46A mutation as well, and likewise,Bc-CspB (with an Ala at position 46)should not be stabilized by the E3R muta-tion. An ion pair between Arg 3 and the C-terminus of Bc-Csp can also be ruled outbecause the extension of Bc-Csp by oneamino acid (as in Bs-CspB), does not affectthe stability. No other negatively chargedside chains approach Arg 3 closer than 6 Å.Clearly, Arg 3 accounts for the additionalelectrostatic stabilization of Bc-Csp and theE3R variant of Bs-CspB, but this stabilization does not seem to involve spe-cific ion pairs. The general stabilizingeffects of single charged residues, possiblyby long range electrostatic interactions,have also been observed in other pro-teins9–11. A part of the electrostatic stabi-lization by Arg 3 could be explained by theabsence of unfavorable repulsive forcesbetween Glu 3 and Glu 66 that exist in wildtype Bs-CspB. While this is a reasonablescenario, the side chains of Glu 3 and Glu66 are disordered in the crystal structure ofBs-CspB5 and therefore, the distancebetween them is unknown.

The molecular environment of Leu 66 inthe high resolution structure of Bc-Csp isshown in Fig. 4. Its side chain packs on the

main chain hydrogen bonds between Val 47, Ser 48 and Val 64,which link the beginning of β-strand 4 with the end of β-strand 5. Leu 66 thus decreases the polarity around thesehydrogen bonds and excludes water molecules as potentialhydrogen bond competitors. This hydrophobic shielding ofhydrogen bonded backbone structure might be the majorcause of stabilization by Leu 66. It is independent of NaCl con-centration (Table 1).

Conclusions and prospectsAttempts to understand the thermostability of proteins rely inlarge part on sequence or structure/sequence comparisonsbetween proteins of thermophilic and mesophilic organ-isms1,12–15. These proteins are typically large and oligomeric orcontain tightly bound cofactors, such as the ferredoxins16, anddenature irreversibly at high temperature. This irreversibilityand the typically large number of sequence differences haveprecluded systematic mutational studies and thermodynamicanalyses as performed here with the two cold shock proteins. Asimilar study was performed with the dimeric DNA bindingprotein HU for which stabilization is linked, among other fac-tors, with improved intermolecular packing17. Recently, wholegenomes of mesophilic and thermophilic bacteria have beenanalyzed to identify changes in the frequencies of particularamino acids18. These global studies are valuable, but cannotgive information as to whether the observed overall drifts inamino acid composition reflect improvements in thermody-namic stability, chemical resistance or solubility at high tem-perature.

Table 1 Stability data for Bc-Csp, Bs-CspB and their variants1

TM2 ∆HD (TM)3 ∆GD

4 ∆∆GD5 ∆GD

4 ∆∆GD5

(70 °C) (70 °C) (70 °C) (70 °C)2 M NaCl 2 M NaCl

Bc-Csp 76.9 ± 0.1 245 ± 5 4.5 – 9.2 –Bc-Csp Q2L 80.9 ± 0.1 242 ± 3 6.8 2.3 10.4 1.2Bc-Csp R3E 59.1 ± 0.1 191 ± 2 -7.0 -11.5 5.0 -4.2Bc-Csp N11S 79.2 ± 0.1 246 ± 3 5.9 1.4 10.1 0.9Bc-Csp Y15F 76.7 ± 0.1 237 ± 2 4.3 -0.2 9.5 0.3Bc-Csp G23Q 74.7 ± 0.1 249 ± 7 3.3 -1.2 7.9 -1.3Bc-Csp S24D 77.8 ± 0.1 258 ± 5 5.4 0.9 10.0 0.8Bc-Csp T31S 77.8 ± 0.1 250 ± 5 5.2 0.7 10.0 0.8Bc-Csp E46A 75.4 ± 0.1 241 ± 3 3.6 -0.9 8.7 -0.5Bc-Csp Q53E 76.1 ± 0.1 246 ± 5 4.1 -0.4 9.2 0.0Bc-Csp V64T 75.1 ± 0.1 244 ± 6 3.4 -1.1 8.4 -0.8Bc-Csp L66E 68.9 ± 0.1 210 ± 3 -0.7 -5.2 3.9 -5.3Bc-Csp 67A 76.9 ± 0.1 247 ± 6 4.6 0.1 8.9 -0.3Bc-Csp R3E/L66E 44.6 ± 0.1 154 ± 1 -16.2 -20.7 -0.4 -9.6

Bs-CspB 53.6 ± 0.1 193 ± 2 -11.3 – 0.8 –Bs-CspB E3R 69.6 ± 0.1 219 ± 2 -0.2 11.1 4.0 3.2Bs-CspB E66L 66.4 ± 0.1 224 ± 2 -2.5 8.8 6.8 6.0Bs-CspB E3R/E66L 74.6 ± 0.1 229 ± 3 2.9 14.2 8.0 7.2

1The thermodynamic data were derived from thermal unfolding transitions performed asdescribed in Fig. 2.2TM, midpoint of the thermal unfolding transition, in °C.3∆HD (TM), enthalpy of unfolding at TM, in kJ mol-1.4∆GD (70 °C), Gibbs free energy of unfolding at 70 °C, in kJ mol-1. The accuracy of the ∆GD (70 °C)values is correlated with TM. For variants with TM values close to 70 °C (between 65 and 75 °C)the accuracy is ∼ 0.3 kJ mol-1. For variants with TM values between 60 and 65 or between 75 and80 °C the accuracy is ∼ 0.5 kJ mol-1.5∆∆GD (70 °C), difference in ∆GD (70 °C) between the variant and the respective wild type pro-tein (Bc-Csp or Bs-CspB).

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Our study of the individual factors that cause incrementalincreases in thermodynamic stability and their structuralinterpretation reveal that the additional stability of a ther-mophilic protein can come from just a few residues at the pro-tein surface. For the cold shock proteins a single chargereversal by a Glu to Arg change led to a remarkable increase inTM of 16 °C. Contrary to what is often assumed, this stabiliza-tion does not seem to be caused by the formation of specificion pairs or ion networks1,19–21, but by a dramatic change in theelectrostatic potential, such that it is converted from net desta-bilizing (as in Bs-CspB) to net stabilizing (as in Bc-Csp)6. Thisagrees well with theoretical concepts that emphasize the gener-al nature of electrostatic interactions12,22,23 and explains thefailure of attempts to convert mesophilic into thermophilicproteins by introducing putative salt bridges that werededuced from a comparison of their crystal structures13,14.

In an evolutionary context, the extra thermostability of Bc-Csp (or the loss of this stability in Bs-CspB) apparentlyarose in a very simple manner from changes at only two posi-tions, one near the N-terminus and the other near the C-ter-minus. Certainly, it would have been difficult to identify thesetwo critical substitutions by sequence or sequence/structurecomparisons alone, because they are accompanied by 10 muta-tions that are unimportant for stability.

These results are very encouraging for the development ofstrategies to increase stability by protein engineering. A fewchanges at surface exposed sites should be sufficient to greatlyincrease stability and in an additive fashion. These sites mustfirst be identified, however, and distinguished from the manyothers which are unimportant for stability. This might beachieved by combining sequence comparisons with computa-tional approaches10,11,24,25. The optimal residues for the select-ed positions can then be identified by techniques of in vitroprotein evolution and selection for optimal stability26–28.

MethodsConstruction and overproduction of protein variants.Variants of Bc-Csp and Bs-CspB were constructed by site-directedmutagenesis of the plasmids pCspBc6 and pCspB329 using theQuikChange kit of Stratagene (La Jolla, CA). The length of theoligonucleotide primers was between 24 and 34 bases with themutant codons placed in the middle. All mutations were con-firmed by sequencing of the entire csp genes. Variants were over-produced by using the T7 RNA polymerase promoter system andpurified as described for wild type Bc-Csp6 and Bs-CspB30 withminor modifications.

Heat induced equilibrium unfolding transitions. Thermalunfolding transitions were measured in 0.1 M Na cacodylate/HCl,pH 7.0, at protein concentrations of 4 µM in the absence or pres-ence of 2 M NaCl. The transitions were monitored by the decreaseof the CD signal at 222.6 nm at 1 nm band width and 1 cm path-length as described6. Heating rates were 60 °C h-1. Transitions were

evaluated using a nonlinear least squares fit according to a two-state model8. In this procedure, a constant value of 4,000 J mol-1 K-1 was used for the change in heat capacity uponunfolding (∆CP). It represents the average of all the values foundin the analyses of the individual thermal transitions. All transitionsshowed similar slopes for the pre-transitional and post-transition-al baselines. Most variants showed TM values between 70 and 80 °Cand therefore extrapolations were not necessary for determiningthe ∆GD values at 70 °C. As a consequence these ∆GD values werefairly insensitive to uncertainties in ∆CP or the baselines.

AcknowledgmentsWe thank the members of our laboratories and the Marahiel laboratory forhelp and discussions as well as C. Brooks III (Scripps Research Institute) and C. Nick Pace (Texas A&M University) for a fruitful exchange of ideas about theelectrostatic stabilization of proteins. This work was supported by grants fromthe Deutsche Forschungsgemeinschaft and the Fonds der ChemischenIndustrie.

Correspondence should be addressed to F.X.S. email: FX.Schmid@uni-bayreuth.de

Received 24 January, 2000; accepted 21 March, 2000.

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