[31] Calorimetric analyses of hyperthermophile proteins

Shriver, John W.; Peters, William B.; Szary, Nicholas M.; Edmondson, Stephen P.

doi:10.1016/s0076-6879(01)34483-x

Cited by 14 publications

(20 citation statements)

References 58 publications

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“…Reversibility of thermal unfolding was demonstrated with repeated scans on the same sample. DSC data were analyzed using an IGOR Pro (Wavemetrics, Oregon) program (IgorDenat, available at http://daffy.uah.edu/thermo) as described elsewhere (29). …”

Section: Methodsmentioning

confidence: 99%

Structure, Stability, and Flexibility of Ribosomal Protein L14e from Sulfolobus solfataricus

et al. 2009

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Ribosomal protein L14e is a component of the large ribosomal subunit in both archaea and eukaryotes. We report here a high resolution NMR solution structure of recombinant L14e and show that the N-terminal 57 residues adopt a classic SH3 fold. The protein contains a tight turn between strands 1 and 2 instead of the typical SH3 RT-loop, indicating that it is unlikely to interact with neighboring ribosomal proteins using the common SH3 site for proline-rich sequences. The remainder of the protein (39 residues) forms a largely extended chain with a short helix which packs onto the surface of the SH3 domain via hydrophobic interactions. It has the potential of adopting an alternative structure to expose a hydrophobic surface for protein-protein interactions in the ribosome without disruption of the SH3 fold. 15 N relaxation data demonstrate that the majority of the Cterminal chain is well defined on the SH3 surface. The globular protein unfolds reversibly with a T m of 102.8 °C at pH 7, making it one of the most stable SH3 domain proteins described to date. The structure of L14e is expected to serve as a model for other members of the L14e family, along with members of the COG2163 group, including L6e and L27e. Interestingly, the N-terminal sequence of L14e shows the greatest similarity of any Sulfolobus protein to the reported N-terminal sequence of Sac8b, a DNA-binding protein reported by Grote et al. (Biochim. Biophys. Acta 873, 405-413 (1986)). The likelihood that L14e and Sac8b are the same protein is discussed. KeywordsArchaea; Sulfolobus; ribosome; protein stabilityThe ribosome is the universal molecular machine which is responsible for translating mRNA into polypeptides and proteins. It is composed of about 80 proteins assembled onto ribosomal RNA to form a 70S particle in prokaryotes (eubacteria and archaea) and a larger 80S particle in eukaryotes. The intact ribosome can be separated into two subunits referred to as small and large: 30S and 50S in prokaryotes, and 40S and 60S in eukaryotes. Although the intact archaeal ribosome subunits are smaller than those in eukaryotes, the archaeal proteins are more similar to those in eukaryotes than eubacteria (1). The structure of the bacterial ribosome has been defined by crystal structures of the 30S and 50S subunits (2-4) and the intact 70S particle (5-8). Crystal structures of the archaeal 50S subunit from Haloarcula (9) and the intact yeast ribosome (10) have been published, along with cryomicroscopy re-constructions of the canine ribosome (11,12 L14e is known to be a protein component of the large ribosome subunit in eukaryotes and Archaea based on 2D gel electrophoresis and sequencing, but the structure and position in the ribosome have not been described. The L14e sequence is conserved in archaea and eukaryotes (including yeast, Drosophila, rat, and human), where the protein is sometimes referred to as L14 (1,(13)(14)(15)(16). L14e shows no homology to the bacterial L14p protein (unfortunately also sometimes referred to as L14) for which a structur...

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Section: Methodsmentioning

confidence: 99%

Structure, Stability, and Flexibility of Ribosomal Protein L14e from Sulfolobus solfataricus

et al. 2009

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“…An alternative method of implementing the noncooperative McGhee–von Hippel model in the analysis of ITC data has been proposed by Shriver and coworkers [37], in which the heat observed by each injection is evaluated by

q_{i} = V_{0} Δ H Δ {[X]}_{b, i} + h

where Δ[ X ] b,i is the change in concentration of bound protein as the result of the i th injection and h is the heat of dilution observed with each injection after saturation of the binding sites at the end of titration [38]. The change in the concentration of bound protein is given by

Δ {[X]}_{b, i} = {[X]}_{b, i} - {[X]}_{b, i - 1} + 0.5 \frac{υ}{V_{0}} ({[X]}_{b, i} + {[X]}_{b, i - 1})

…”

Section: Cooperativity Of Long-chain Macromolecules With Multiple Binmentioning

confidence: 99%

Analysis of Cooperativity by Isothermal Titration Calorimetry

Brown

2009

IJMS

125

107

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Cooperative binding pervades Nature. This review discusses the use of isothermal titration calorimetry (ITC) in the identification and characterisation of cooperativity in biological interactions. ITC has broad scope in the analysis of cooperativity as it determines binding stiochiometries, affinities and thermodynamic parameters, including enthalpy and entropy in a single experiment. Examples from the literature are used to demonstrate the applicability of ITC in the characterisation of cooperative systems.

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“…For presentation purposes, the baselines of both scans have been flattened by removing the DC p of unfolding as described. 100 The data were fit by non-linear regression to a two-state model with unfolding of the dimer to two random-coil chains to obtain a T m of 92. Crystal Structure of Sso10a described to date.…”

Section: Antiparallel Coiled-coil Dna-binding Proteinsmentioning

confidence: 99%

“…99 Differential scanning calorimetry DSC was performed on a MicroCal VP-DSC with extended temperature-range capability using standard procedures. 100 The protein was dialyzed extensively against the desired buffer and degassed using the apparatus supplied by MicroCal with the calorimeter. The protein, at a concentration of 0.9 mg/ml, was scanned at 1.5 deg.…”

Section: Circular Dichroismmentioning

confidence: 99%