Optimized variants of the cold shock protein from in vitro selection: structural basis of their high thermostability

The bacterial cold shock proteins (Csp) are widely used as models for the experimental and computational analysis of protein stability. In a previous study, in vitro evolution was employed to identify strongly stabilizing mutations in Bs-CspB from Bacillus subtilis. The best variant found by this approach contained the mutations M1R, E3K and K65I, which raised the midpoint of thermal unfolding of Bs-CspB from 53.8 degrees C to 83.7 degrees C, and increased the Gibbs free energy of stabilization by 20.9 kJ mol(-1). Another selected variant with the two mutations A46K and S48R was stabilized by 11.1 kJ mol(-1). To elucidate the molecular basis of these stabilizations, we determined the crystal structures of these two Bs-CspB variants. The mutated residues are generally well ordered and provide additional stabilizing interactions, such as charge interactions, additional hydrogen bonds and improved side-chain packing. Several mutations improve the electrostatic interactions, either by the removal of unfavorable charges (E3K) or by compensating their destabilizing interactions (A46K, S48R). The stabilizing mutations are clustered at a contiguous surface area of Bs-CspB, which apparently is critically important for the stability of the beta-barrel structure but not well optimized in the wild-type protein.     

Interaction of nucleolar phosphoprotein B23 with nucleic acids

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.     

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.     

Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.     

Specificity of DNA binding and dimerization by CspE from Escherichia coli

The CspE protein from Escherichia coli K12 is a single-stranded nucleic acid-binding protein that plays a role in chromosome condensation in vivo. We report here that CspE binds to single-stranded DNA containing 6 or more contiguous dT residues with high affinity (K(D) < 30 nM). The interactions are predominantly through base-specific contacts. When an oligonucleotide contains fewer than 6 contiguous dT residues, the CspE interactions with single-stranded DNA are primarily electrostatic. The minimal length of single-stranded DNA to which CspE binds in a salt-resistant manner is eight nucleotides. We also show that CspE exists as a dimer in solution. We present a possible mechanism to explain the role of CspE in chromosome condensation in vivo by CspE binding to distant DNA regions in the chromosome and dimerizing, thereby condensing the intervening DNA.     

Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein

The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential.     

Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding

The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.     

Complete amino acid sequence of three reptile lysozymes

To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.     

Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding

The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase

Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.     

Proteins from the prokaryotic nucleoid. Interaction of nucleic acids with the 15 kDa Escherichia coli histone-like protein H-NS

The interaction between nucleic acids and Escherichia coli H-NS, an abundant 15 kDa histone-like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy. Intrinsic fluorescence studies showed that the single Trp residue of H-NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers. Binding of H-NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)-poly(dT) and poly(dC)-poly(dG) it can be estimated that an H-NS dimer in 1.5 x SSC binds DNA with an apparent Ka approximately equal to 1.1 x 10(4) M-1.bp-1. H-NS binds to double-stranded DNA with a higher affinity than the more abundant histone-like protein NS(HU) and, unlike NS, prefers double-stranded to single-stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.     

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.     

A spin probe approach for measuring the nucleic acid affinity of gene 32 protein.

A novel and fast procedure for determining by electron spin resonance the affinity of proteins for nucleic acids is described. The assay makes use of nitroxide radicals which are covalently bound to various polynuleotides to the extent of one probe per 75 to 100 nucleotides. As a test example gene 32 protein was used, a protein known to interact with single stranded nucleic acids. Competition experiments with unlabeled nucleic acids made it feasible to directly monitor the gene 32 protein affinity for different nucleic acids. It was observed that DNA single strands are not necessarily favored by this protein for preferential binding. The experimental data also suggest that the difference in the binding constants for most of the complexes is remarkably large; for instance, (dT)_n binds at least 3 to 4 orders of magnitude better to gene 32 protein than (dA)_n.     

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.     

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.