Contribution of the 30/36 Hydrophobic Contact at the C-Terminus of the α-Helix to the Stability of the Ubiquitin Molecule

Thomas, Susan T.; Makhatadze, George I.

doi:10.1021/bi0000418

Cited by 28 publications

(33 citation statements)

References 58 publications

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“…This pH was selected because at this pH the transition temperature for the WT protein is 60°C (12) and thus more stable variants will have the unfolding transition completed before reaching the upper limit (110°C) of the operating temperature range of the DSC instrument. The reference temperature was set at 50°C because it is an average temperature of unfolding of all ubiquitin variants studied at pH 3.0, and thus the extrapolation error over the entire set of variants is minimal.…”

Section: Resultsmentioning

confidence: 99%

“…Overexpression of the ubiquitin variants was done in the BL21(DE3) strain of Escherichia coli as described (11). Each variant was purified to apparent homogeneity as described (12). For reasons discussed previously (13), all described ubiquitin variants contained R63K substitution.…”

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confidence: 99%

“…The protein concentration for ubiquitin mutants varied between 1.0 and 3.0 mg͞ml. Detailed procedures of the sample preparation for the DSC experiment were the same as described (12,15). The partial molar heat capacity of the protein, C p,pr (T), was obtained from the experimentally measured apparent heat capacity difference between the sample (containing protein solution) and reference (containing corresponding buffer solution) cells, ⌬C p app , using the following expression:…”

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confidence: 99%

“…Protein concentration was measured spectrophotometrically by using a known extinction coefficient for the WT ubiquitin, 1cm,280nm 0.1% ϭ 0.149 (11)(12)(13). This extinction coefficient was used for all but G35W ubiquitin variants because none of those amino acid substitutions introduced residues that absorb in the far UV range.…”

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confidence: 99%

“…Analysis of the heat capacity profiles was done by using nonlinear regression routine NLREG and in-house written scripts (15). Individual curves for a given ubiquitin variant were fit to a two-state transition model with the heat capacities of the native and the unfolded states, the enthalpy of unfolding, ⌬H cal , heat capacity of unfolding, ⌬C p , and transition temperature, T m , as independent variables (12). The standard thermodynamic functions under reference conditions were calculated as:…”

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confidence: 99%

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Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

Thomas

Loladze

Makhatadze

2001

Proc. Natl. Acad. Sci. U.S.A.

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The C position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C position in the ␣-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physicochemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C position in an ␣-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala> Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the 1 H-15 N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (r ‫؍‬ 0.85) with the statistical distribution of the residues at the C position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (r ‫؍‬ 0.85) and the calculated hydration of the peptide backbone (r ‫؍‬ 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C position of the C-capping box in ␣-helices. This propensity scale is useful for protein secondary structure predictions and protein design.T he first basic structural motif of globular proteins proposed was the ␣-helix (1). The structure of the ␣-helix is characterized by hydrogen bonding patterns between amide hydrogen bond donors and carbonyl oxygen acceptors of residues situated four apart in the sequence. This pattern of hydrogen bonding, however, implies that the four initial amide hydrogen bond donors and the four final carbonyl oxygen hydrogen bond acceptors do not have hydrogen bonding partners. The potential effect of this is fraying of the helix ends. In 1988, Richardson and Richardson (2) as well as Presta and Rose (3) analyzed statistical preferences of the amino acid residues at the ends of ␣-helices and revealed the existence of specific capping interactions at both the N and C termini, which compensate for the unsatisfied hydrogen bonds and thus prevent the ends from fraying. According to Aurora et al. (4) and Harper and Rose (5), amino acid residues in helices and their flanking residues can be labeled as:where numbered residues have helical backbone dihedral angles ( ϭ Ϫ64 Ϯ 7 o ; ϭ Ϫ41 Ϯ 7 o ). Ncap with dihedrals ϭ Ϫ94 Ϯ 15 o and ϭ 167 Ϯ 5 o (5) and Ccap with dihedrals ϷϪ81 o and ϷϪ2 o (6) are boundary residues that belong both to the helix and the adjacent turn. Residues at the ...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

Thomas

Loladze

Makhatadze

2001

Proc. Natl. Acad. Sci. U.S.A.

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Thermal coefficients of the methyl groups within ubiquitin

et al. 2012

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Physiological processes such as protein folding and molecular recognition are intricately linked to their dynamic signature, which is reflected in their thermal coefficient. In addition, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross-correlated relaxation between dipolar couplings of two CH bonds within ubiquitin. Spanning a temperature range from 275 to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N-terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy.

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Both helical propensity and side‐chain hydrophobicity at a partially exposed site in α‐helix contribute to the thermodynamic stability of ubiquitin

Loladze

Makhatadze

2004

Proteins

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Improving helical propensity of residues was proposed as one of the approaches to increase protein stability. Here the contribution of the helix propensity and hydrophobicity of residues at partially buried positions of alpha-helix to the stability of a model protein-ubiquitin- is explored. Thermodynamic stabilities of 13 ubiquitin variants with substitutions at a partially buried helical residue were measured by differential scanning calorimetry. It was found that the dynamic range of stabilities for different amino acid residues at this partially buried position is 3 times larger than that expected based on helical propensity alone. Correlation analysis shows that both helical propensity and hydrophobicity are important in defining the relative stabilities of the studied ubiquitin variants. These results provide experimental evidence that partially buried positions are potentially useful sites for engineering proteins with enhanced thermostability.

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Contribution of the 30/36 Hydrophobic Contact at the C-Terminus of the α-Helix to the Stability of the Ubiquitin Molecule

Cited by 28 publications

References 58 publications

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

Thermal coefficients of the methyl groups within ubiquitin

Both helical propensity and side‐chain hydrophobicity at a partially exposed site in α‐helix contribute to the thermodynamic stability of ubiquitin

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