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Udo Heinemann - One of the best experts on this subject based on the ideXlab platform.
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rna single strands bind to a conserved surface of the major Cold Shock Protein in crystals and solution
RNA, 2012Co-Authors: Rolf Sachs, Udo Heinemann, Klaas E A Max, Jochen BalbachAbstract:Bacterial Cold Shock Proteins (CSPs) regulate the cellular response to temperature downshift. Their general principle of function involves RNA chaperoning and transcriptional antitermination. Here we present two crystal structures of Cold Shock Protein B from Bacillus subtilis (Bs-CspB) in complex with either a hexanucleotide (5′-UUUUUU-3′) or heptanucleotide (5′-GUCUUUA-3′) single-stranded RNA (ssRNA). Hydrogen bonds and stacking interactions between RNA bases and aromatic sidechains characterize individual binding subsites. Additional binding subsites which are not occupied by the ligand in the crystal structure were revealed by NMR spectroscopy in solution on Bs-CspB·RNA complexes. Binding studies demonstrate that Bs-CspB associates with ssDNA as well as ssRNA with moderate sequence specificity. Varying affinities of oligonucleotides are reflected mainly in changes of the dissociation rates. The generally lower binding affinity of ssRNA compared to its ssDNA analog is attributed solely to the substitution of thymine by uracil bases in RNA.
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t rich dna single strands bind to a preformed site on the bacterial Cold Shock Protein bs cspb
Journal of Molecular Biology, 2006Co-Authors: Klaas E A Max, Jochen Balbach, Markus Zeeb, Ralf Bienert, Udo HeinemannAbstract:Bacterial Cold Shock Proteins (CSPs) are involved in cellular adaptation to Cold stress. They bind to single-stranded nucleic acids with a KD value in the micro- to nanomolar range. Here we present the structure of the Bacillus subtilis CspB (Bs-CspB) in complex with hexathymidine (dT6) at a resolution of 1.78 A. Bs-CspB binds to dT6 with nanomolar affinity via an amphipathic interface on the Protein surface. Individual binding subsites interact with single nucleobases through stacking interactions and hydrogen bonding. The sugar-phosphate backbone and the methyl groups of the thymine nucleobases remain solvent exposed and are not contacted by Protein groups. Fluorescence titration experiments monitoring the binding of oligopyrimidines to Bs-CspB reveal binding preferences at individual subsites and allow the design of an optimised heptapyrimidine ligand, which is bound with sub-nanomolar affinity. This study reveals the stoichiometry and sequence determinants of the binding of single-stranded nucleic acids to a preformed site on Bs-CspB and thus provides the structural basis of the RNA chaperone and transcription antitermination activities of the CSP.
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two exposed amino acid residues confer thermostability on a Cold Shock Protein
Nature Structural & Molecular Biology, 2000Co-Authors: Dieter Perl, Udo Heinemann, Uwe Mueller, Franz X SchmidAbstract:Thermophilic organisms produce Proteins of exceptional stability. To understand Protein thermostability at the molecular level we studied a pair of Cold Shock Proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two Proteins differ in sequence at 12 positions, two surface-exposed residues are responsible 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 a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic Protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a Protein.
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thermal stability and atomic resolution crystal structure of the bacillus caldolyticus Cold Shock Protein
Journal of Molecular Biology, 2000Co-Authors: U Mueller, Franz X Schmid, Dieter Perl, Udo HeinemannAbstract: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.
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crystal structure of cspa the major Cold Shock Protein of escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Hermann Schindelin, Masayori Inouye, Weining Jiang, Udo HeinemannAbstract:The major Cold Shock Protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The Protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic Proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major Cold Shock Protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a Protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial Cold Shock Proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common Cold Shock domain.
Masayori Inouye - One of the best experts on this subject based on the ideXlab platform.
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selective mrna degradation by polynucleotide phosphorylase in Cold Shock adaptation in escherichia coli
Journal of Bacteriology, 2001Co-Authors: Kunitoshi Yamanaka, Masayori InouyeAbstract:Upon Cold Shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific Cold Shock Proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-Cold Shock Protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon Cold Shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon Cold Shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in Cold Shock adaptation.
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characterization of escherichia coli cspe whose product negatively regulates transcription of cspa the gene for the major Cold Shock Protein
Molecular Microbiology, 1999Co-Authors: Weonhye Bae, Sangita Phadtare, Konstantin Severinov, Masayori InouyeAbstract:Escherichia coli contains nine members of the CspA Protein family from CspA to CspI. To elucidate the cellular function of CspE, we constructed a ΔcspE strain. CspE is highly produced at 37°C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary-phase cells into the fresh medium at 37°C. This is consistent with the ΔcspE phenotype of the longer growth lag period after dilution. The Protein synthesis patterns of the ΔcspE strain and the wild-type strain were compared using two-dimensional gel electrophoresis. In the ΔcspE strain, the synthesis of a number of Proteins at 37°C was found to be altered and cspA was derepressed. The derepression of cspA in the ΔcspE strain was at the level of transcription in a promoter-independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after Cold Shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the Cold box, a putative repressor binding site on the cspA mRNA. In a cell-free Protein synthesis system using S-30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37°C, probably by interacting with the transcription elongation complex at the cspA Cold box region.
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solution nmr structure and backbone dynamics of the major Cold Shock Protein cspa from escherichia coli evidence for conformational dynamics in the single stranded rna binding site
Biochemistry, 1998Co-Authors: Wenqing Feng, Masayori Inouye, Roberto Tejero, Diane E Zimmerman, Gaetano T MontelioneAbstract:The major Cold-Shock Protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding Protein that is produced in response to Cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114−5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Cα, C‘, HN, Hα, and side chain Cβ resonance assignme...
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cspa the major Cold Shock Protein of escherichia coli negatively regulates its own gene expression
Journal of Bacteriology, 1997Co-Authors: W Bae, Pamela G. Jones, Masayori InouyeAbstract:When the gene for CspA, the major Cold Shock Protein of Escherichia coli, was disrupted by a novel positive/negative selection method, the deltacspA cells did not show any discernible growth defect at either 37 or 15 degrees C. By two-dimensional gel electrophoresis, total Protein synthesis was analyzed after temperature downshift in the deltacspA strain. The production of the CspA homologs CspB and CspG increased, and the duration of their expression was prolonged, suggesting that both CspB and CspG compensate for the function of CspA in the absence of CspA during Cold Shock adaptation. Interestingly, the production of the 159-base 5'-untranslated region (5'-UTR) of cspA from the chromosomal cspA::cat gene, detected by primer extension, failed to be repressed after Cold Shock. When an independent system to produce CspA was added to the deltacspA strain, the 5'-UTR production for the cspA::cat gene was significantly reduced compared to that of the deltacspA strain. By examining the expression of translationally fused cspA and cspB genes to lacZ in the deltacspA strain, it was found that cspA is more strongly regulated by CspA than cspB is. We showed that the increased expression of the 5'-UTR of the cspA mRNA in the deltacspA strain occurred mainly at the level of transcription and, to a certain extent, at the level of mRNA stabilization. The mRNA stabilization in the deltacspA strain was observed for other mRNAs, supporting the notion that CspA functions as an mRNA chaperone to destabilize secondary structures in mRNAs.
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cspa the major Cold Shock Protein of escherichia coli is an rna chaperone
Journal of Biological Chemistry, 1997Co-Authors: Weining Jiang, Yan Hou, Masayori InouyeAbstract:Abstract CspA, the major Cold-Shock Protein of Escherichia coli, is dramatically induced during the Cold-Shock response. The amino acid sequence of CspA shows 43% identity to the “Cold-Shock domain” of the eukaryotic Y-box Protein family, which interacts with RNA and DNA to regulate their functions. Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis. A minimal concentration of CspA at 2.7 × 10−5 M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10−4 M) in Cold-Shocked cells. No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding. When the 142-base 5′-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5′-untranslated region. These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases. We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature. Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.
Jochen Balbach - One of the best experts on this subject based on the ideXlab platform.
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rna single strands bind to a conserved surface of the major Cold Shock Protein in crystals and solution
RNA, 2012Co-Authors: Rolf Sachs, Udo Heinemann, Klaas E A Max, Jochen BalbachAbstract:Bacterial Cold Shock Proteins (CSPs) regulate the cellular response to temperature downshift. Their general principle of function involves RNA chaperoning and transcriptional antitermination. Here we present two crystal structures of Cold Shock Protein B from Bacillus subtilis (Bs-CspB) in complex with either a hexanucleotide (5′-UUUUUU-3′) or heptanucleotide (5′-GUCUUUA-3′) single-stranded RNA (ssRNA). Hydrogen bonds and stacking interactions between RNA bases and aromatic sidechains characterize individual binding subsites. Additional binding subsites which are not occupied by the ligand in the crystal structure were revealed by NMR spectroscopy in solution on Bs-CspB·RNA complexes. Binding studies demonstrate that Bs-CspB associates with ssDNA as well as ssRNA with moderate sequence specificity. Varying affinities of oligonucleotides are reflected mainly in changes of the dissociation rates. The generally lower binding affinity of ssRNA compared to its ssDNA analog is attributed solely to the substitution of thymine by uracil bases in RNA.
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recognition of t rich single stranded dna by the Cold Shock Protein bs cspb in solution
Nucleic Acids Research, 2006Co-Authors: Markus Zeeb, Klaas E A Max, Ulrich Weininger, Christian Low, Heinrich Sticht, Jochen BalbachAbstract:Cold Shock Proteins (CSP) belong to the family of single-stranded nucleic acid binding Proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT7) by NMR spectroscopy. BsCspB reveals an almost invariant conformation when bound to dT7 with only minor reorientations in loop b1–b2 and b3–b4 and of few aromatic side chains involved in base stacking. Binding studies of Protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15 N NMR relaxation and H/D exchange experiments revealed that binding of dT7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire Protein and especially of loop b3–b4.
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t rich dna single strands bind to a preformed site on the bacterial Cold Shock Protein bs cspb
Journal of Molecular Biology, 2006Co-Authors: Klaas E A Max, Jochen Balbach, Markus Zeeb, Ralf Bienert, Udo HeinemannAbstract:Bacterial Cold Shock Proteins (CSPs) are involved in cellular adaptation to Cold stress. They bind to single-stranded nucleic acids with a KD value in the micro- to nanomolar range. Here we present the structure of the Bacillus subtilis CspB (Bs-CspB) in complex with hexathymidine (dT6) at a resolution of 1.78 A. Bs-CspB binds to dT6 with nanomolar affinity via an amphipathic interface on the Protein surface. Individual binding subsites interact with single nucleobases through stacking interactions and hydrogen bonding. The sugar-phosphate backbone and the methyl groups of the thymine nucleobases remain solvent exposed and are not contacted by Protein groups. Fluorescence titration experiments monitoring the binding of oligopyrimidines to Bs-CspB reveal binding preferences at individual subsites and allow the design of an optimised heptapyrimidine ligand, which is bound with sub-nanomolar affinity. This study reveals the stoichiometry and sequence determinants of the binding of single-stranded nucleic acids to a preformed site on Bs-CspB and thus provides the structural basis of the RNA chaperone and transcription antitermination activities of the CSP.
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single stranded dna binding of the Cold Shock Protein cspb from bacillus subtilis nmr mapping and mutational characterization
Protein Science, 2003Co-Authors: Markus Zeeb, Jochen BalbachAbstract:Rapid adaptation to changes in the environment is essential for the survival of bacteria under extreme conditions such as chemical stress and heat or Cold Shock. A reduction in growth temperature elicits a Cold-Shock response, in the course of which a number of distinct essential Proteins are induced (Jones and Inouye 1994), in particular members of the family of small Cold-Shock Proteins (CSPs). CSPs are ubiquitous Proteins found in psychrotrophic, mesophilic, thermophilic, and hyperthermophilic bacteria (Jones et al. 1987; Hebraud and Potier 1999; Phadtare et al. 1999). The induction originates from an increase in transcription of their genes (Jiang et al. 1993) and from the stabilization of their mRNA (Brandi et al. 1996; Goldenberg et al. 1996). The basal concentrations and levels of induction vary strongly among the CSPs due to their different functions during cell division or during the stationary phase (Yamanaka et al. 1998; Brandi et al. 1999). CSPs stimulate the transcription of Cold-Shock inducible genes (Jones et al. 1992) and facilitate the initiation of translation by destabilizing nonproductive secondary structures in mRNA at low temperature (Jiang et al. 1997) without apparent sequence specificity (Lopez et al. 1999, 2001; Lopez and Makhatadze 2000) and are therefore regarded as “RNA chaperones.” B. subtilis contains three homologous CSPs (CspB, CspC, CspD), which can complement each other in vivo (Graumann et al. 1997). The structure of the small acidic Protein CspB (67 residues, Fig. 1 ▶) has been solved both in crystal and in solution, and revealed a very similar Protein backbone conformation to CspA from Escherichia coli (Schindelin et al. 1993, 1994; Schnuchel et al. 1993; Newkirk et al. 1994; Feng et al. 1998). CspB comprises a five-stranded antiparallel β-barrel with perpendicularly arranged strands of subdomain I (β-strands 1, 2, and 3) and subdomain II (β-strands 4 and 5). It belongs to the OB-fold superfamily (Murzin 1993), which also includes ribosomal Proteins S1 and S17, and translational initiation factor IF1 of E. coli (Sette et al. 1997; Draper and Reynaldo 1999). CSPs recognize DNA and RNA with the two conserved binding motifs RNP1 and RNP2, which comprise amino acids K13-V20 and V26-H29 in CspB, respectively. Investigations by mutagenesis and gel retardation experiments revealed that several aromatic and basic residues within the RNP motifs constitute the putative nucleic acid binding surface (Schroder et al. 1995). Until now, no structure of a Cold-Shock Protein in complex with a single-stranded nucleic acid has been reported. CspB shows low sequence specificity with a preference for thymidine and uridine rich stretches (Schroder et al. 1995; Lopez et al. 1999, 2001; Phadtare and Inouye 1999). U-rich regions can be found upstream of the promotor in the unusually long 5′-untranslated region (5′-UTR) of the CspB mRNA, and T-rich stretches can be located at factor-independent transcription termination signals (Richardson 1993; Henkin 1996; Phadtare and Inouye 1999). Figure 1. Ribbon drawing of the Cold-Shock Protein B from B. subtilis. The three-dimensional structure consists of two perpendicularly arranged subdomains (I: β-strands β1–β3, II: β-strands β4 and β5), which ... Domains homologous to the prokaryotic CSPs are the CSDs of eukaryotic Y-box transcription factors and nucleic acid-binding Proteins, such as the telomere end binding Protein (Graumann and Marahiel 1998; Horvath et al. 1998). CSDs are also involved in RNA binding of ribosomal Proteins (Draper and Reynaldo 1999; Nakagawa et al. 1999) or of mitochondrial Y-box Proteins (Pelletier et al. 2000). In the present article we characterized the structural basis of CspB binding to a 25-mer ssDNA fragment termed Y-Box25, containing the Y-box core motif ATTGG, which is a cis-element in the promoter region of mammalian MHC II genes (Didier et al. 1988). Oligodeoxynucleotides containing Y-Box25 are the standard substrates of CspB used in previous studies (Schnuchel et al. 1993; Graumann and Marahiel 1994; Schroder et al. 1995; Schindler et al. 1999). Seventeen CspB residues involved in ssDNA binding were identified by line broadening of their backbone amides in a titration experiment followed by heteronuclear 2D NMR. They include eight residues of the proposed RNP motifs. Interestingly, seven amino acid residues in two loop regions that are not part of the classical RNP motifs show substantial changes in the chemical shifts of their backbone resonances upon binding. They report on remote conformational rearrangements in the nucleoProtein complex. The contributions of residues, identified by NMR, to the Gibbs free energy of binding were quantitatively characterized by equilibrium and kinetic fluorescence quenching experiments of 15 Protein variants containing single amino acid substitutions. All examined aromatic and positively charged residues are necessary for a tight binding of Y-Box25. In addition, conformational restriction in loop β3–β4 decreases the binding affinity significantly. Reduced binding correlates with increased dissociation, rather than association, rate constants compared to the wild-type Protein. This might be of importance for the “RNA chaperone” function of CspB.
Franz X Schmid - One of the best experts on this subject based on the ideXlab platform.
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specificity of the initial collapse in the folding of the Cold Shock Protein
Journal of Molecular Biology, 2006Co-Authors: Christine Magg, Georg Holtermann, Jan Kubelka, Elisha Haas, Franz X SchmidAbstract:The two-state folding reaction of the Cold Shock Protein from Bacillus caldolyticus (Bc-Csp) is preceded by a rapid chain collapse. A fast shortening of intra-Protein distances was revealed by Forster resonance energy transfer (FRET) measurements with Protein variants that carried individual pairs of donor and acceptor chromophores at various positions along the polypeptide chain. Here we investigated the specificity of this rapid compaction. Energy transfer experiments that probed the stretching of strand β2 and the close approach between the strands β1 and β2 revealed that the β1–β2 hairpin is barely formed in the collapsed form, although it is native-like in the folding transition state of Bc-Csp. The time course of the collapse could not be resolved by pressure or temperature jump experiments, indicating that the collapsed and extended forms are not separated by an energy barrier. The co-solute (NH4)2SO4 stabilizes both native Bc-Csp and the collapsed form, which suggests that the large hydrated SO42- ions are excluded from the surface of the collapsed form in a similar fashion as they are excluded from folded Bc-Csp. Ethylene glycol increases the stability of Proteins because it is excluded preferentially from the backbone, which is accessible in the unfolded state. The collapsed form of Bc-Csp resembles the unfolded form in its interaction with ethylene glycol, suggesting that in the collapsed form the backbone is still accessible to water and small molecules. Our results thus rule out that the collapsed form is a folding intermediate with native-like chain topology. It is better described as a mixture of compact conformations that belong to the unfolded state ensemble. However, some of its structural elements are reminiscent of the native Protein.
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stabilization of the Cold Shock Protein cspb from bacillus subtilis by evolutionary optimization of coulombic interactions
Journal of Molecular Biology, 2005Co-Authors: Michael Wunderlich, Andreas Martin, Franz X SchmidAbstract: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.
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the folding transition state of the Cold Shock Protein is strongly polarized
Journal of Molecular Biology, 2004Co-Authors: Maria M Garciamira, Daniel Boehringer, Franz X SchmidAbstract:Abstract The Cold Shock Protein CspB from Bacillus subtilis consists of a three-stranded (β1–β3) and a two stranded (β4–β5) sheet, which form a closed β barrel structure. CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 °C). The transition state of folding is native-like, as judged by the Tanford β T value of ≥0.9. By using a mutational approach and Φ value analysis, we find that the folding transition state of CspB is energetically polarized. Despite the high β T value, most Φ values are low. Values close to 1 were found for only a few residues, particularly in strand β1 (Lys5, Val6, Lys7, Asn10). The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands β1 and β2. Lys5 and Val6 in β1 interact with residues in β4, and their high Φ values indicate that an energetic linkage between β1 and β4 and thus between the two sheets exists already in the transition state. We compared our experimental Φ values with theoretical predictions of the folding pathway of Cold Shock Proteins. Several of them suggest that the entire first sheet is formed in the transition state, and some identify the β1–β4 pairing as a crucial step in folding. Alternative paths that involve formation of the second sheet and β3–β5 pairing reactions were, however, suggested as well. The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure. They did not identify the key residues with the high Φ values within these structural elements.
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rapid collapse precedes the fast two state folding of the Cold Shock Protein
Journal of Molecular Biology, 2004Co-Authors: Christine Magg, Franz X SchmidAbstract:The Cold Shock Protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-Protein distances during folding by Forster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the Protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this Protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.
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the effects of ionic strength on Protein stability the Cold Shock Protein family
Journal of Molecular Biology, 2002Co-Authors: Brian N Dominy, Dieter Perl, Franz X Schmid, Charles L BrooksAbstract:Continuum electrostatic models are used to examine in detail the mechanism of Protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type Cold Shock Proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these Proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of Protein stability toward salt is correlated with thermostability in the Cold Shock Protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the Protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed Cold Shock Protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic Proteins.
Hermann Schindelin - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of cspa the major Cold Shock Protein of escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Hermann Schindelin, Masayori Inouye, Weining Jiang, Udo HeinemannAbstract:The major Cold Shock Protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The Protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic Proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major Cold Shock Protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a Protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial Cold Shock Proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common Cold Shock domain.
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universal nucleic acid binding domain revealed by crystal structure of the b subtilis major Cold Shock Protein
Nature, 1993Co-Authors: Hermann Schindelin, Moharned A. Marahiel, Udo HeinemannAbstract:THE Cold-Shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature. It leads to the increased production of the major Cold-Shock Proteins, CS7.4 and CspB, respectively1–3. CS7.4 is a transcriptional activator of two genes4,5. CS7.4 and CspB share 43 per cent sequence identity with the nucleic acid-binding domain of the eukaryotic gene-regulatory Y-box factors6. This Cold-Shock domain is conserved from bacteria to man7 and contains the RNA-binding RNP1 sequence motif8. As a prototype of the Cold-Shock domain, the structure of CspB has been determined here from two crystal forms. In both, CspB is present as an antiparallel five-stranded β-barrel. Three consecutive β-strands, the central one containing the RNP1 motif, create a surface rich in aromatic and basic residues that are presumably involved in nucleic acid binding. Preferential binding of CspB to single-stranded DNA is observed in gel retardation experiments.
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universal nucleic acid binding domain revealed by crystal structure of the b subtilis major Cold Shock Protein
Nature, 1993Co-Authors: Hermann Schindelin, Mohamed A Marahiel, Udo HeinemannAbstract:The Cold-Shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature. It leads to the increased production of the major Cold-Shock Proteins, CS7.4 and CspB, respectively. CS7.4 is a transcriptional activator of two genes. CS7.4 and CspB share 43 per cent sequence identity with the nucleic acid-binding domain of the eukaryotic gene-regulatory Y-box factors. This Cold-Shock domain is conserved from bacteria to man and contains the RNA-binding RNP1 sequence motif. As a prototype of the Cold-Shock domain, the structure of CspB has been determined here from two crystal forms. In both, CspB is present as an antiparallel five-stranded beta-barrel. Three consecutive beta-strands, the central one containing the RNP1 motif, create a surface rich in aromatic and basic residues that are presumably involved in nucleic acid binding. Preferential binding of CspB to single-stranded DNA is observed in gel retardation experiments.