Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Balbach, Jochen; Seip, Stephan; Kessler, Horst; Scharf, Matthias; Kashani‐Poor, Noushin; Engels, Joachim W.

doi:10.1002/(sici)1097-0134(19981101)33:2<285::aid-prot11>3.0.co;2-g

Cited by 12 publications

(7 citation statements)

References 49 publications

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“…That is, in mutants M 1 and M 2 , only one disulfide bond is kept, and in mutant M 3 , both disulfide bonds are removed. In our theoretical study, the native structures of these three mutants are obtained by energy minimization based on the wild-type native structure; this takes into consideration in experiment that their native structures were observed to be similar to the wild type (28). The energy minimization is realized by using Amber software (version 7.0) (39) after the replacements of the related residues (17).…”

Section: Model and Methodsmentioning

confidence: 99%

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Effects of Disulfide Bonds on Folding Behavior and Mechanism of the β-Sheet Protein Tendamistat

Qin

Jian

Wang

2006

Biophysical Journal

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Tendamistat, a small disulfide-bonded beta-sheet protein, and its three single/double-disulfide mutants are investigated by using a modified Gō-like model, aiming to understand the folding mechanism of disulfide-bonded protein as well as the effects of removal of disulfide bond on the folding process. Our simulations show that tendamistat and its two single-disulfide mutants are all two-state folders, consistent with the experimental observations. It is found that the disulfide bonds as well as three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat. Without these interactions, their two-state behaviors become unstable and the predictions of the model are inconsistent with experiments. In addition, the effect of disulfide bonds on the folding process are studied by comparing the wild-type tendamistat and its two mutants; it is found that the removal of either of the C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and loss of structure in the unfolded state, and the effect of the former is stronger than that of the later. These simulation results are in good agreement with experiments and, thus, validate our model. Based on the same model, the detailed folding pathways of the wild-type tendamistat and two mutants are studied, and the effect of disulfide bonds on the folding kinetics are discussed. The obtained results provide a detailed folding picture of these proteins and complement experimental findings. Finally, the folding nuclei predicted to be existent in this protein tendamistat as well as its mutants are firstly identified in this work. The positions of the nucleus are consistent with those argued in experimental studies. Therefore, a nucleation/growth folding mechanism that can explain the two-state folding manner is clearly characterized. Moreover, the effect by the removal of each disulfide bond on the folding thermodynamics and dynamics can also be well interpreted from their influence on the folding nucleus. The implementation of this work indicates that the modified Gō-like model really describes the folding behavior of protein tendamistat and could be used to study the folding of other disulfide-bonded proteins.

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Section: Model and Methodsmentioning

confidence: 99%

“…It is an all-b-sheet protein with 74 amino acids and has two disulfide bonds (25,26). It has been well studied in a number of experiments (16,17,19,20,(27)(28)(29)(30). It was found that protein tendamistat exhibits a two-state folding behavior that has not been found in any other disulfidebonded proteins up to now.…”

Section: Introductionmentioning

confidence: 99%

Effects of Disulfide Bonds on Folding Behavior and Mechanism of the β-Sheet Protein Tendamistat

Qin

Jian

Wang

2006

Biophysical Journal

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“…1,2 In practice, however, this desirable effect is sometimes foiled by strain or reduced dynamics imposed on the native state, especially in the case of artificially introduced disulfide bonds. [3][4][5] Even though disulfide bridges are one of the best understood factors stabilizing the native structure of a protein, [6][7][8] their kinetic role in protein folding is not comprehensively understood. Disulfide bonds can lead to the persistence of partially structured or at least conformationally restricted regions in otherwise unfolded polypeptides.…”

Section: Introductionmentioning

confidence: 99%

Influence of the Internal Disulfide Bridge on the Folding Pathway of the CL Antibody Domain

Feige

Hagn

Esser

et al. 2007

Journal of Molecular Biology

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“…Despite many studies involving removal of natural disulfide links (Schwartz et al 1987; Pace et al 1988; Eigenbrot et al 1990; Cooper et al 1992; Ikeguchi et al 1992; Kuroki et al 1992; Vogl et al 1995; Bonander et al 2000; Klink et al 2000) and insertion of novel ones (Sauer et al 1986; Wells and Powers 1986; Pantoliano et al 1987; Villafranca et al 1987; Pjura et al 1990; Takagi et al 1990; Matsumura and Matthews 1991; Clarke and Fersht 1993; Clarke et al 1995; Hinck et al 1996; Johnson et al 1997; Futami et al 2000), including those made by chemical modification (Goldenberg and Creighton 1983; Lin et al 1984; Ueda et al 1985), our understanding of the mechanism of stabilization of proteins by cross‐links is far from complete. Loop length alone does not fully account for observed changes in denaturation free energy or entropy (Zhang et al 1994; Vogl et al 1995; Balbach et al 1998) Disulfide mutants show decreased as well as increased stabilities, and their denaturation thermodynamic parameters do not support the Doig and Williams model (Matsumura and Matthews 1991; Johnson et al 1997).…”

mentioning

confidence: 99%

“…Steric and electrostatic effects of the amino acid side‐chains that replace the disulfide bond are believed to influence protein stability (Vogl et al 1995; Balbach et al 1998). Theoretical studies indicate that destabilization by a cross‐link can occur through reduction of configurational entropy of the folded state (Tidor and Karplus 1993).…”

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Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

et al. 2001

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Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T m ), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (⌬⌬G d 50°C ‫ס‬ −4 kcal/mole; ⌬T m ‫ס‬ −22°C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (⌬⌬G d 50°C ‫ס‬ −3 kcal/mole; ⌬T m ‫ס‬ −11°C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys 44 -Asp 45 peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (⌬⌬G d 50°C ‫ס‬ −1 kcal/mole; ⌬T m ‫ס‬ +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (⌬⌬G d 50°C ‫ס‬ +1 kcal/mole; ⌬T m ‫ס‬ +17°C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.Keywords: Disulfide-bond; cross-link; protein stability; differential scanning calorimetry; denaturation; folding Supplemental material: See www.proteinscience.org.The conformational stability of a protein is important to its function. Certain diseases such as Alzheimer's, prion, and cystic fibrosis, are associated with misfolded, unfolded, or aggregated proteins (Kelly 1996;Harper and Lansbury, Jr. 1997;Horwich and Weissman 1997, Qu et al. 1997). Much has been characterized regarding contributions to protein stability of hydrogen bonds, i...

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Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Cited by 12 publications

References 49 publications

Effects of Disulfide Bonds on Folding Behavior and Mechanism of the β-Sheet Protein Tendamistat

Effects of Disulfide Bonds on Folding Behavior and Mechanism of the β-Sheet Protein Tendamistat

Influence of the Internal Disulfide Bridge on the Folding Pathway of the CL Antibody Domain

Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

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