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The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog.

Cuneo MJ, Tian Y, Allert M, Hellinga HW - BMC Struct. Biol. (2008)

Bottom Line: We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C).The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains.The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute for Biological Structure and Design and the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, 27710, USA. mjc18@duke.edu

ABSTRACT

Background: Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability.

Results: We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 A resolution. We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C). The tteRBP has essentially the identical backbone conformation (0.41 A RMSD of 235/271 Calpha positions and 0.65 A RMSD of 270/271 Calpha positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities.

Conclusion: The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

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Amino acid sequence comparison of tteRBP and ecRBP. Clustal-W amino acid sequence alignment of tteRBP and ecRBP. Amino acids which are not conserved are in bold type and underlined, amino acids that are conserved but not identical are in bold type (charge inversions are scored as non-conservative here). Core, boundary or surface classification of amino acids is shown below the aligned residues.
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Figure 1: Amino acid sequence comparison of tteRBP and ecRBP. Clustal-W amino acid sequence alignment of tteRBP and ecRBP. Amino acids which are not conserved are in bold type and underlined, amino acids that are conserved but not identical are in bold type (charge inversions are scored as non-conservative here). Core, boundary or surface classification of amino acids is shown below the aligned residues.

Mentions: ORF tte0206 in the T. tengcongensis genome sequence [29] was postulated to be a ribose-binding protein homolog (tteRBP) based on its sequence similarity to the known E. coli RBP (57% identity, 76% similarity) (Figure 1) and its position within a putative operon containing ORFs homologous to ABC transporters characteristic of solute transport. The DNA for ORF tte0206, lacking a putative periplasmic signal sequence [30] (residues 1–39), was amplified from T. tengcongensis genomic DNA by the polymerase chain reaction. The resulting DNA fragment was cloned into a pET21a vector in-frame with a C-terminal hexa-histidine tag preceded by a glycine-serine linker. The nucleotide sequence was confirmed by DNA sequencing of the resulting vector. Over-expression of tte0206 produced ~30 mg of pure protein per liter of medium, which was purified by immobilized metal affinity chromatography followed by gel filtration chromatography. tteRBP eluted from the gel filtration column as a broad peak immediately following the void volume of the column (data not shown). For subsequent crystallization and characterization of tteRBP fractions of the broad peak from the gel filtration column, that were consistent with monomeric tteRBP, (fractions with a calculated hydrodynamic radius of 30 kDa ± 15 kDa) were pooled and concentrated to ~15 mg/mL (see Materials and Methods).


The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog.

Cuneo MJ, Tian Y, Allert M, Hellinga HW - BMC Struct. Biol. (2008)

Amino acid sequence comparison of tteRBP and ecRBP. Clustal-W amino acid sequence alignment of tteRBP and ecRBP. Amino acids which are not conserved are in bold type and underlined, amino acids that are conserved but not identical are in bold type (charge inversions are scored as non-conservative here). Core, boundary or surface classification of amino acids is shown below the aligned residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315655&req=5

Figure 1: Amino acid sequence comparison of tteRBP and ecRBP. Clustal-W amino acid sequence alignment of tteRBP and ecRBP. Amino acids which are not conserved are in bold type and underlined, amino acids that are conserved but not identical are in bold type (charge inversions are scored as non-conservative here). Core, boundary or surface classification of amino acids is shown below the aligned residues.
Mentions: ORF tte0206 in the T. tengcongensis genome sequence [29] was postulated to be a ribose-binding protein homolog (tteRBP) based on its sequence similarity to the known E. coli RBP (57% identity, 76% similarity) (Figure 1) and its position within a putative operon containing ORFs homologous to ABC transporters characteristic of solute transport. The DNA for ORF tte0206, lacking a putative periplasmic signal sequence [30] (residues 1–39), was amplified from T. tengcongensis genomic DNA by the polymerase chain reaction. The resulting DNA fragment was cloned into a pET21a vector in-frame with a C-terminal hexa-histidine tag preceded by a glycine-serine linker. The nucleotide sequence was confirmed by DNA sequencing of the resulting vector. Over-expression of tte0206 produced ~30 mg of pure protein per liter of medium, which was purified by immobilized metal affinity chromatography followed by gel filtration chromatography. tteRBP eluted from the gel filtration column as a broad peak immediately following the void volume of the column (data not shown). For subsequent crystallization and characterization of tteRBP fractions of the broad peak from the gel filtration column, that were consistent with monomeric tteRBP, (fractions with a calculated hydrodynamic radius of 30 kDa ± 15 kDa) were pooled and concentrated to ~15 mg/mL (see Materials and Methods).

Bottom Line: We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C).The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains.The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute for Biological Structure and Design and the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, 27710, USA. mjc18@duke.edu

ABSTRACT

Background: Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability.

Results: We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 A resolution. We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C). The tteRBP has essentially the identical backbone conformation (0.41 A RMSD of 235/271 Calpha positions and 0.65 A RMSD of 270/271 Calpha positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities.

Conclusion: The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

Show MeSH
Related in: MedlinePlus