Mechanism of Protein Stabilization by Disulfide Bridges: Calorimetric Unfolding Studies on Disulfide-deficient Mutants of the α-Amylase Inhibitor Tendamistat

Vogl, Thomas; Brengelmann, Ralf; Hinz, Hans‐Jürgen; Scharf, Matthias; Lötzbeyer, Martin; Engels, Joachim W.

doi:10.1006/jmbi.1995.0632

Cited by 58 publications

(70 citation statements)

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“…Our model showed that the disulfide bonds as well as the three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat (79); without either interaction, the two-state behaviors become unstable or the folding pathway changes significantly. The simulation based on the modified model showed that tendamistat and its two singledisulfide mutants are all two-state folders, consistent with the experimental observations (76,79). By comparing the folding behaviors of the wild-type tendamistat with its two mutants, it was found that the removal of either C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and the loss of structure in the unfolded state; and the effect of the former is stronger than that of the latter, as shown in Fig.…”

Section: The Rules Played By Disulfide Bonds In the Folding Process Osupporting

confidence: 70%

“…It is an allb-sheet protein with 74 amino acids and has two disulfide bonds, C11-C27 and C45-C73. There are extensive experimental studies on this protein (72)(73)(74)(75)(76)(77)(78)(79). As a brief summary of these works, tendamistat exhibits a two-state folding behavior that is unique among all the disulfide bonded proteins discovered so far, and this behavior sustains if either disulfide bond is removed (76).…”

Section: The Rules Played By Disulfide Bonds In the Folding Process Omentioning

confidence: 99%

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Protein folding simulations: From coarse‐grained model to all‐atom model

Jian

Wang

et al. 2009

IUBMB Life

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SummaryProtein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL, the development of discrete MD and its application in studying the a-b conformational conversion and oligomer assembling process, and the modeling of metal ion involved protein folding.

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Section: The Rules Played By Disulfide Bonds In the Folding Process Osupporting

confidence: 70%

Section: The Rules Played By Disulfide Bonds In the Folding Process Omentioning

confidence: 99%

Protein folding simulations: From coarse‐grained model to all‐atom model

Jian

Wang

et al. 2009

IUBMB Life

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“…As a result, it has been suggested that secreted enzymes such as ␣-amylases may be stabilized by maintaining disulfide bridges (4). Removal of disulfide bridges has been shown to cause loss of enzyme activity (36) and stability (35,37), and the formation and reduction of disulfide bridges may function as an on-off switch to regulate enzyme activity in response to stress (4).…”

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confidence: 99%

Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase

et al. 2005

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The cold-adapted ␣-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with ␤-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.

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“…A detailed thermodynamic characterization of tendamistat wild-type and its disulfide variants by differential scanning calorimetry, 15 as well as a folding study using fluorescence-and CD-spectroscopy, 16 surprisingly showed that opening of the small segment (C11-C27) caused a larger destabilization in terms of both enthalpy and entropy than opening the large segment (C45-C73), which caused a smaller decrease of stability.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1998

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Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the alpha-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by 13C(alpha) heteronuclear NOE experiments with carbon in natural abundance. 15N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15N resonance assignment was achieved by a 15N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the beta-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel beta-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat.

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Mechanism of Protein Stabilization by Disulfide Bridges: Calorimetric Unfolding Studies on Disulfide-deficient Mutants of the α-Amylase Inhibitor Tendamistat

Cited by 58 publications

References 0 publications

Protein folding simulations: From coarse‐grained model to all‐atom model

Protein folding simulations: From coarse‐grained model to all‐atom model

Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase

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

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