Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Borén, Kristina; Andersson, Peter; Larsson, Marie; Carlsson, Uno

doi:10.1016/s0167-4838(98)00283-0

Cited by 34 publications

(23 citation statements)

References 24 publications

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“…Initially, it can be pointed out that the transition curves we obtained by recording the various parameters did not coincide ( Figure 3C), which is evidence that A23C/L203C ox also undergoes sequential unfolding. The first event in unfolding of A23C/L203C ox entailed inactivation that occurred concomitant to the change in the CD band at 270 nm (C m ) 1.7 M), which implies that, at this stage, the active-site region was conformationally rearranged, and there was a concurrent loss of the asymmetric tertiary structure surrounding the aromatic residues, especially the Trp residues (41,42). Inasmuch as the unfolding transition revealed by internal Trp fluorescence occurred at somewhat higher GuHCl concentrations (C m ) 1.8 M) than the corresponding inactivation and CD transitions, the latter FIGURE 5: A thermodynamic double-mutant cycle for determining the coupling energy between Ala23 and Leu203 in the native state.…”

Section: Discussionmentioning

confidence: 99%

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

2002

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To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/ L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

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Section: Discussionmentioning

confidence: 99%

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

2002

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“…Borén et al and Mårtensson et al observed that the negative ellipticity measured in the far-UV was larger for the intermediate than for the native state of BCA II and HCA II, respectively; they interpreted this increase as being due to loss of interference from aromatic side chains in the symmetric environment of a molten globule. 676,720 The large negative ellipticity indicates the presence of extensive β-structure in the molten globule. When the concentration of GuHCl increased, CA II denatured further, and the negative ellipticity decreased.…”

Section: Significant Secondary Structurementioning

confidence: 99%

“…When the concentration of GuHCl increased, CA II denatured further, and the negative ellipticity decreased. 676,720 Jonasson et al used site-directed mutagenesis to remove systematically Trp residues from the Cys206Ser mutant of HCA II in order to identify their individual contributions to fluorescence. 675 They denatured the protein and observed refolding by monitoring the fluorescence of the Trp residues.…”

Section: Significant Secondary Structurementioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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“…Low pH or addition of alcohol or other denaturants can induce partially folded conformations in some proteins [4][5][6]. Trifluoroethanol (TFE) is known to induce non-native partially folded states in proteins and it is often used as an agent for inducing and/or stabilizing the secondary structure (helix), but destabilizing the specific tertiary interactions of the native protein [3,[7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Comparative studies on trifluoroethanol (TFE) state of a thermophilic α-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes

Asghari

Khajeh

Ranjbar

et al. 2004

International Journal of Biological Macromolecules

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Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Cited by 34 publications

References 24 publications

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Comparative studies on trifluoroethanol (TFE) state of a thermophilic α-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes

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