Thermal Unfolding of the DNA-binding Protein Sso7d from the HyperthermophileSulfolobus solfataricus

Knapp, Stefan; Karshikoff, Andrey; Berndt, Kurt D.; Christova, P.; Атанасов, Б.; Ladenstein, Rudolf

doi:10.1006/jmbi.1996.0701

Cited by 94 publications

(110 citation statements)

References 48 publications

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“…3) revealed a comparable change in ellipticity over this wavelength range upon unfolding. Although not typical of ␤-sheet proteins, the native state spectra in Figure 3 are characteristic of SH3 domains (Lim et al 1994;Viguera et al 1994;Knapp et al 1996;Renzoni et al 1996;Bousquet et al 2000;Okishio et al 2003). In the absence of ␣-helical structure, CD bands in the region of 225 nm have been attributed to aromatic side chains (Schmid 1989).…”

Section: Resultsmentioning

confidence: 99%

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

2004

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Under native conditions, apocytochrome b 5 exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a ⌬T m of 8.3°C, a ⌬C m of 1.5 M urea, and a ⌬⌬G°of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the ∼6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b 5 suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.Keywords: protein structure/folding; stability and mutagenesis Supplemental material: see www.proteinscience.orgIn the fashion of many ligand-binding proteins, the watersoluble domain of rat hepatic cytochrome b 5 undergoes induced refolding upon association with its heme cofactor. In the holoprotein state, the three-dimensional structure is well represented by a narrow ensemble of conformations. As illustrated in Figure 1, the fold contains six helices and five strands (Mathews et al. 1979). The heme is fastened in its cavity by coordination bonds ligating the Fe ion to the imidazole N atoms of His 39 (end of ␣2) and His 63 (beginning of ␣4). Two hydrophobic regions, termed core 1 and core 2, are also apparent in the cytochrome structure. The residues of core 1 are located in the central part of the amino acid sequence; they establish most of the protein-heme contacts and therefore play a functional role. In contrast, the residues comprising core 2 are found within the N-and C-terminal segments. Core 2 does not require the heme Abbreviations: ⌬␦, change in chemical shift in ppm; bp, base pair; CD, circular dichroism; DQF-COSY, double-quantum-filtered correlated spectroscopy; EbE1, chimeric protein whose N-and C-terminal stretches are composed of residues from PsaE and whose intervening residues come from cytochrome b 5 ; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropylthio-␤-D-galactoside;...

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Section: Resultsmentioning

confidence: 99%

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

2004

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“…Examples in support of the contention include the observation that native (methylated) and recombinant (unmethylated) chromatin protein Sac7d, a member of the Sul7d family from S. acidocaldarius, differ by ϳ6°C in melting point temperature (Tm) (65). However, no significant differences were detected in thermal stability between the methylated and unmenthylated forms of Sso7d, a highly close homolog of Sac7d from S. solfataricus (66). Taking advantage of the availability of the aKMT deletion mutant, in which the level of protein methylation was substantially lower than that in the parental strain, we compared the thermal stability of the cellular proteins from the two strains.…”

Section: Discussionmentioning

confidence: 99%

aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism

Chu

Zhu

Chen

et al. 2016

Molecular & Cellular Proteomics

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Protein methylation is believed to occur extensively in creanarchaea. Recently, aKMT, a highly conserved crenarchaeal protein lysine methyltransferase, was identified and shown to exhibit broad substrate specificity in vitro. Here, we have constructed an aKMT deletion mutant of the hyperthermophilic crenarchaeon Sulfolobus islandicus. The mutant was viable but showed a moderately slower growth rate than the parental strain under nonoptimal growth conditions. Consistent with the moderate effect of the lack of aKMT on the growth of the cell, expression of a small number of genes, which encode putative functions in substrate transportation, energy metabolism, transcriptional regulation, stress response proteins, etc, was differentially regulated by more than twofold in the mutant strain, as compared with that in the parental strain. Analysis of the methylation of total cellular protein by mass spectrometry revealed that methylated proteins accounted for ϳ2/3 (1,158/1,751) and ϳ1/3 (591/ 1,757) of the identified proteins in the parental and the mutant strains, respectively, indicating that there is extensive protein methylation in S. islandicus and that aKMT is a major protein methyltransferase in this organism. No significant sequence preference was detected at the sites of methylation by aKMT. Methylated lysine residues, when visible in the structure, are all located on the surface of the proteins. The crystal structure of aKMT in complex with S-adenosyl-L-methionine (SAM) or S-adenosyl homocysteine (SAH) reveals that the protein consists of four ␣ helices and seven ␤ sheets, lacking a substrate recognition domain found in PrmA, a bacterial homolog of aKMT, in agreement with the broad substrate specificity of aKMT. Our results suggest that aKMT may serve a role in maintaining the methylation status of cellular proteins required for the efficient growth of the organism under certain non-optimal conditions. Molecular & Cellular

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“…Under these scanning conditions the cuvette was in thermal equilibrium with the cell holder. 41 A cuvette with 1 mm path length was used for measurements on Btk and Itk SH3; for Tec SH3, a cuvette with 1 cm path length was used. The pH of the sample was measured before and after the scans and was found to be constant within 0.1 pH units.…”

Section: Spectroscopymentioning

confidence: 99%

“…Small proteins, like SH3 domains, unfold usually in a two-state transition without detectable intermediates. 40,41 Very often, experimental conditions can be found where the unfolding transition of domains is a reversible process. Furthermore, the comparison of the SH3-like folding motif from the extremely thermophilic archaeon Sulfolobus with its mesophilic counterparts is expected to give insight into the question of how proteins are adapted to different thermal environments.…”

Section: Introductionmentioning

confidence: 99%

“…[42][43][44] The thermal unfolding transition was studied by circular dichroism (CD) spectroscopy in the acidic and neutral pH region, as well as by differential scanning calorimetry (DSC) in the case of Btk-SH3. The thermodynamic data were compared to those of the hyperthermostable β-barrel protein Sso7d 41 and the SH3 domain in ␣-spectrin. 40 …”

Section: Introductionmentioning

confidence: 99%

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Thermal unfolding of small proteins with SH3 domain folding pattern

et al. 1998

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The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.

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Thermal Unfolding of the DNA-binding Protein Sso7d from the HyperthermophileSulfolobus solfataricus

Cited by 94 publications

References 48 publications

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism

Thermal unfolding of small proteins with SH3 domain folding pattern

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Thermal Unfolding of the DNA-binding Protein Sso7d from the HyperthermophileSulfolobus solfataricus

Cited by 94 publications

References 48 publications

Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism

Thermal unfolding of small proteins with SH3 domain folding pattern

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Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture