Bacterial cold-shock proteins.

Members of a family of small cold-shock proteins (CSPs) are induced during bacterial cell response to a temperature decrease. Here we review available data about the structure, molecular properties, mechanism of induction and possible functions of CSPs. CSPs preferentially bind single-stranded RNA and DNA and appear to play an important role in cell physiology under both normal and cold-shock conditions. Although the function of CSPs in cold-shock adaptation has not yet been elucidated in detail, a number of experimental evidences suggests that CSPs bind messenger RNA (mRNA) and regulate ribosomal translation, rate of mRNA degradation and termination of transcription.

Molecular characterization and tissue distribution of a novel member of the S100 family of EF-hand proteins.

We have isolated from a human prostate cDNA library a cDNA encoding a novel member of the S100 family of EF-hand proteins. The encoded 99-amino acid protein, designated S100Z, is capable of interacting with another member of the family, S100P. S100Z cDNA was cloned into a bacterial expression system, and the S100Z protein was purified to homogeneity from bacterial lysates by a combination of hydrophobic column and gel-filtration chromatography. Direct amino acid sequencing of the 20 N-terminal amino acids confirmed that the sequence of the recombinant protein is identical to the sequence deduced from the cDNA. Low-resolution structural data have been obtained using circular dichroism and fluorescence spectroscopies, and equilibrium analytical centrifugation. These results show that S100Z is a dimeric, predominantly alpha-helical protein. Addition of calcium to a solution of S100Z changes the fluorescence intensity of the protein, indicating that S100Z is capable of binding calcium ions. Analysis of the calcium-binding isotherm indicates the existence of two calcium-binding sites with apparent affinities on the order of 5 x 10(6) and 10(2) M(-1). Binding of calcium results in conformational changes and exposure of hydrophobic surfaces on the protein. Using a PCR-based assay, we have detected differences in the expression level of S100Z mRNA in various tissues. The highest levels were found in spleen and leukocytes. S100Z gene expression appears to be deregulated in some tumor tissues, compared to expression in their normal counterparts.

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C' position of the C-capping box of alpha-helices.

The C' position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C' position in the alpha-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physico-chemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C' position in an alpha-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C' position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala>Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the (1)H-(15)N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (r = 0.85) with the statistical distribution of the residues at the C' position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (r = 0.85) and the calculated hydration of the peptide backbone (r = 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C' position of the C-capping box in alpha-helices. This propensity scale is useful for protein secondary structure predictions and protein design.

Heat capacity changes upon burial of polar and nonpolar groups in proteins.

In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution.

Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the T base content and position within the template.

The cold shock protein CspB from Bacillus subtilis binds T-based single-stranded DNA (ssDNA) with high affinity (Lopez, M. M., Yutani, K., and Makhatadze, G. I. (1999) J. Biol. Chem. 274, 33601-33608). In this paper we report the results of CspB interactions with non-homogeneous ssDNA templates containing continuous and non-continuous stretches of T bases. The analysis of CspB-ssDNA interactions was performed using fluorescence spectroscopy, analytical centrifugation and isothermal titration calorimetry. We show that (i) there is a strong correlation between the CspB affinity and stoichiometry and the T content in the oligonucleotide that is independent of which other bases are incorporated into the sequence of ssDNA; (ii) the binding properties of CspB to ssDNA templates with continuous or non-continuous stretches of T bases with similar T content is very similar, and (iii) the mechanism of interaction between CspB and the T-based non-homogeneous ssDNA is mainly through the bases (a stretch of three T bases located in the middle of the ssDNA templates makes the binding independent of the ionic strength). The biological relevance of these results to the role of CspB as an RNA chaperone is discussed.

Energetics of target peptide binding by calmodulin reveals different modes of binding.

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.

To charge or not to charge?

The ability to engineer proteins with increased thermostability will profoundly broaden their practical applications. Recent experimental results show that optimization of charge-charge interactions on the surface of proteins can be a useful strategy in the design of thermostable enzymes. Results also indicate a possibility that such optimized interactions provide structural determinants for enhanced stability of proteins from thermophilic organisms. In this article, the general strategy for design of thermostable proteins and perspectives for future studies are discussed.

Mapping the energy surface for the folding reaction of the coiled-coil peptide GCN4-p1.

The energy surface for the folding/unfolding reactions of the homodimeric coiled-coil peptide M2V GCN4-p1, a 33-residue segment comprising the leucine zipper domain of the transcriptional activator GCN4, was mapped by equilibrium and kinetic methods. Circular dichroism (CD) spectroscopy was used to monitor the urea-induced unfolding reaction at a series of temperatures and temperature-induced unfolding at a series of urea concentrations. A global analysis of the urea- and temperature-induced equilibrium unfolding data provides strong support for a two-state mechanism. The absence of a detectable population of intermediate states is also consistent with differential scanning calorimetry and thermal CD melts as a function of peptide concentration. Furthermore, a global analysis of stopped-flow CD kinetic data is consistent with a kinetic two-state mechanism as well. The urea dependence of the apparent folding and unfolding rate constants at a series of temperatures reveals that the activation enthalpy and entropy for unfolding in the absence of denaturant are both significantly greater than those for the refolding reaction. Although the unfolding barrier is dominated by the activation enthalpy, the activation entropy dominates the refolding barrier. The relative magnitudes of the urea dependence of the unfolding and refolding rate constants indicate that 55-65% of the surface area is buried in the transition state. The activation parameters imply a partially organized transition state and are consistent with a previous model in which the pair of C-terminal heptad repeats are docked in a coiled-coil-like motif [Zitzewitz et al. (2000) J. Mol. Biol. 296, 1105-1116].

Measuring protein thermostability by differential scanning calorimetry.

Differential scanning calorimetry (DSC) the method of choice to study the conformational stability of biological macromolecules and proteins in particular. This unit presents step-by-step protocols for DSC, including sample preparation and interpretation of the results in the simplest cases as well as calibration of the apparatus and maintenance of DSC cells. Various general experimental considerations, including possible pitfalls and errors, are also discussed.

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies.

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.

Contribution of the 30/36 hydrophobic contact at the C-terminus of the alpha-helix to the stability of the ubiquitin molecule.

The contribution of the hydrophobic contact in the C-capping motif of the alpha-helix to the thermodynamic stability of the ubiquitin molecule has been analyzed. For this, 16 variants of ubiquitin containing the full combinatorial set of four nonpolar residues Val, Ile, Leu, and Phe at C4 (Ile30) and C' ' (Ile36) positions were generated. The secondary structure content as estimated using far-UV circular dichroism (CD) spectroscopy of all but Phe variants at position 30 did not show notable changes upon substitutions. The thermodynamic stability of these ubiquitin variants was measured using differential scanning calorimetry, and it was shown that all variants have lower stability as measured by decreases in the Gibbs energy. Since in some cases the decrease in stability was so dramatic that it rendered an unfolded protein, it was therefore concluded that, despite apparent preservation of the secondary structure, the 30/36 hydrophobic contact is essential for the stability of the ubiquitin molecule. The decrease in the Gibbs energy in many cases was found to be accompanied by a large (up to 25%) decrease in the enthalpy of unfolding, particularly significant in the variants containing Ile to Leu substitutions. This decrease in enthalpy of unfolding is proposed to be primarily the result of the perturbed packing interactions in the native state of the Ile --> Leu variants. The analysis of these data and comparison with effects of similar amino acid substitutions on the stability of other model systems suggest that Ile --> Leu substitutions cannot be isoenergetic at the buried site.

Major cold shock proteins, CspA from Escherichia coli and CspB from Bacillus subtilis, interact differently with single-stranded DNA templates.

The family of bacterial major cold shock proteins is characterized by a conserved sequence of 65-75 amino acid residues long which form a three-dimensional structure consisting of five beta-sheets arranged into a beta-barrel topology. CspA from Escherichia coli and CspB from Bacillus subtilis are typical representative members of this class of proteins. The exact biological role of these proteins is still unclear; however, they have been implicated to possess ssDNA-binding activity. In this paper, we report the results of a comparative quantitative analysis of ssDNA-binding activity of CspA and CspB. We show that in spite of high homology on the level of primary structure and very similar three-dimensional structures, CspA and CspB have different ssDNA-binding properties. Both proteins preferentially bind polypyrimidine ssDNA templates, but CspB binds to the T-based templates with one order of magnitude higher affinity than to U- or C-based ssDNA, whereas CspA binds T-, U- or C-based ssDNA with comparable affinity. They also show similarities and differences in their binding to ssDNA at high ionic strength. The results of these findings are related to the chemical structure of DNA bases.

Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA.

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.

Primary structure determinants of the pH- and temperature-dependent aggregation of thioredoxin.

Thioredoxins are small proteins found in all living organisms. We have previously reported that Chlamydomonas reinhardtii thioredoxin h exhibited differences both in its absorption spectrum and its aggregation properties compared to thioredoxin m. In this paper, we demonstrate, by site-directed mutagenesis, that the particularity of the absorption spectrum is linked to the presence of an additional tryptophan residue in the h isoform. The pH and temperature dependence of the aggregation of both thioredoxins has been investigated. Our results indicate that the aggregation of TRX is highly dependent on pH and that the differences between the two TRX isoforms are linked to distinct pH dependencies. We have also analyzed the pH and temperature dependence of 12 distinct variants of TRX engineered by site-directed mutagenesis. The results obtained indicate that the differences in the hydrophobic core of the two TRX isoforms do not account for the differences of aggregation. On the other hand, we show the importance of His-109 as well as the second active site cysteine, Cys-39 in the aggregation mechanism.

Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface.

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.

Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition.

CspB is a small acidic protein of Bacillus subtilis, the induction of which is increased dramatically in response to cold shock. Although the exact functional role of CspB is unknown, it has been demonstrated that this protein binds single-stranded deoxynucleic acids (ssDNA). We addressed the question of the effect of base composition on the CspB binding to ssDNA by analyzing the thermodynamics of CspB interactions with model oligodeoxynucleotides. Combinations of four different techniques, fluorescence spectroscopy, gel shift mobility assays, isothermal titration calorimetry, and analytical ultracentrifugation, allowed us to show that: 1) CspB can preferentially bind poly-pyrimidine but not poly-purine ssDNA templates; 2) binding to T-based ssDNA template occurs with high affinity (K(d(25 degrees C)) approximately 42 nM) and is salt-independent, whereas binding of CspB to C-based ssDNA template is strongly salt-dependent (no binding is observed at 1 M NaCl), indicating large electrostatic component involved in the interactions; 3) upon binding each CspB covers a stretch of 6-7 thymine bases on T-based ssDNA; and 4) the binding of CspB to T-based ssDNA template is enthalpically driven, indicating the possible involvement of interactions between aromatic side chains on the protein with the thymine bases. The significance of these results with respect to the functional role of CspB in the bacterial cold shock response is discussed.

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.

Heat capacity change for ribonuclease A folding.

The change in heat capacity deltaCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, deltaCp = 1.15+/-0.08 kcal mol(-1) K(-1). Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.

Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.

Cold denaturation of ubiquitin.

Temperature induced unfolding of bovine ubiquitin in solutions with different concentrations of guanidinium hydrochloride (GdmCl) has been measured using differential scanning calorimetry. It has been shown that at high concentrations of GdmCl the ubiquitin molecule can undergo both heat and cold induced denaturation. Analysis of the enthalpy of unfolding of ubiquitin in the presence of GdmCl shows a good agreement with the thermodynamic denaturant binding model. The unfolding Gibbs energy is found to change linearly with guanidine concentration up to zero denaturant concentration.