Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB fromBacillus subtilis

Schindler, Thomas; Perl, Dieter; Graumann, Peter L.; Sieber, Volker; Marahiel, Mohamed A.; Schmid, Franz X.

doi:10.1002/(sici)1097-0134(19980301)30:4<401::aid-prot7>3.0.co;2-l

Cited by 55 publications

(16 citation statements)

References 34 publications

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“…However, too few hydrophobic residues on the surface may reduce the overall stability of proteins because hydrophobic interactions are the major driving force of protein stability (26). Even surface hydrophobic residues improve protein stability (89, 101). Thus, weighting various energetic terms differently leads to different outcomes.…”

Section: Computational Assessment Of Designed Proteinsmentioning

confidence: 99%

Energy Functions in De Novo Protein Design: Current Challenges and Future Prospects

Yang

Zhan

et al. 2013

Annu. Rev. Biophys.

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In the past decade, a concerted effort to successfully capture specific tertiary packing interactions produced specific three-dimensional structures for many de novo designed proteins that are validated by nuclear magnetic resonance and/or X-ray crystallographic techniques. However, the success rate of computational design remains low. In this review, we provide an overview of experimentally validated, de novo designed proteins and compare four available programs, RosettaDesign, EGAD, Liang-Grishin, and RosettaDesign-SR, by assessing designed sequences computationally. Computational assessment includes the recovery of native sequences, the calculation of sizes of hydrophobic patches and total solvent-accessible surface area, and the prediction of structural properties such as intrinsic disorder, secondary structures, and three-dimensional structures. This computational assessment, together with a recent community-wide experiment in assessing scoring functions for interface design, suggests that the next-generation protein-design scoring function will come from the right balance of complementary interaction terms. Such balance may be found when more negative experimental data become available as part of a training set.

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Section: Computational Assessment Of Designed Proteinsmentioning

confidence: 99%

Energy Functions in De Novo Protein Design: Current Challenges and Future Prospects

Yang

Zhan

et al. 2013

Annu. Rev. Biophys.

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“…The construction and purification of the variants with single Phe f Ala replacements at positions 15, 17, and 27 have been described (36,37). Csp from B. caldolyticus was purified according to ref 36. Heat-Induced Equilibrium Unfolding Transitions.…”

Section: Materials Cspb From B Subtilis Was Overexpressed Inmentioning

confidence: 99%

“…Here we used the pressure-jump technique to determine the thermodynamic activation parameters ∆G q , ∆H q , ∆C p q , and ∆S q of the folding reactions of wild-type Bs-CspB and of the Phe15Ala, Phe17Ala, and Phe27Ala variants. These partially exposed Phe residues are located in -strands 2 and 3 (43) and are important for the stability of the folded protein (37). Here we asked whether these Phe residues are also important for the kinetics of folding and how they contribute to the energetics of the activated state of folding.…”

Section: Pressure-induced Unfolding Andmentioning

confidence: 99%

Microsecond Folding of the Cold Shock Protein Measured by a Pressure-Jump Technique

et al. 1999

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A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.

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“…We hoped that the HDXMS on the different forms of the protein in solution would help to determine whether the dimer in solution is consistent with the solved crystal structure [47]. Furthermore, as the aromatic residues of the RNP1 and RNP2 motifs have also been shown to have an important role in protein stability [48], it is interesting to assess the conformational dynamics of dimeric Bc CspΔ36–37 in comparison with its monomeric counterparts through this experimental strategy.…”

Section: Resultsmentioning

confidence: 99%

Unusual dimerization of a BcCsp mutant leads to reduced conformational dynamics

et al. 2017

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Cold shock proteins (Csp) constitute a family of ubiquitous small proteins that act as RNA-chaperones to avoid cold-induced termination of translation. All members contain two subdomains composed of 2 and 3 β-strands respectively, which are connected by a hinge loop and fold into a β-barrel. Bacillus caldolyticus Csp (BcCsp) is one of the most studied members of the family in terms of its folding, function and structure. This protein has been described as a monomer in solution, although a recent crystal structure showed dimerization via domain swapping (DS). In contrast, other cold shock proteins of the same fold are known to dimerize in a non-swapped arrangement. Hypothesizing that reducing the size of the hinge loop may promote swapping as in several other DS proteins with different folds we deleted two residues from these region (BcCspΔ36–37), leading to a protein in monomer-dimer equilibrium with similar folding stability to that of the wild-type. Strikingly, the crystal structure of BcCspΔ36–37 revealed a non-swapped dimer with its interface located at the nucleic acid-binding surface, showing that the deletion led to structural consequences far from the perturbation site. Concomitantly, circular dichroism experiments on BcCspΔ36–37 demonstrated that binding of the oligonucleotide hexathymidine disrupts the dimer. Additionally, HDXMS shows a protective effect on the protein structure upon dimerization, where the resulting interactions between ligand-binding surfaces in the dimer reduced the extent of exchange throughout the whole protein. Our work provides evidence of the complex interplay between conformational dynamics, deletions and oligomerization within the Csp protein family.

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Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB fromBacillus subtilis

Cited by 55 publications

References 34 publications

Energy Functions in De Novo Protein Design: Current Challenges and Future Prospects

Energy Functions in De Novo Protein Design: Current Challenges and Future Prospects

Microsecond Folding of the Cold Shock Protein Measured by a Pressure-Jump Technique

Unusual dimerization of a BcCsp mutant leads to reduced conformational dynamics

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