The molten globular intermediate form in the folding pathway of human carbonic anhydrase B

Jagannadham, Medicherla V.; Balasubramanian, D.

doi:10.1016/0014-5793(85)80396-3

Cited by 54 publications

(20 citation statements)

References 19 publications

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“…Thus, at this concentration of GdmCl, HCA partially unfolds to its molten-globule state. This result is consistent with the earlier report (37). However, in contrast to HCA, BCA possesses a significant extent of tertiary structure in 1.5 M GdmCl in the absence of EDTA as shown in Fig.…”

Section: ␣-Crystallin Binds To Molten-globulesupporting

confidence: 93%

“…In other words, removal of Zn 2ϩ ion alters the stability of the enzyme and its unfolding properties, increasing the propensity of the enzyme to adopt the molten-globule state. The molten-globule states of both human and bovine carbonic anhydrases have been known for long time (37)(38)(39). However, the propensity of its formation, as modulated by the Zn 2ϩ ion, has not been recognized earlier.…”

Section: ␣-Crystallin Binds To Molten-globulementioning

confidence: 99%

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Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Krishnan

Raman

Rao

1996

Journal of Biological Chemistry

103

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␣-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of ␣-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60°C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn 2؉ , by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. ␣-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the ␣-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins.␣-Crystallin is a multimeric protein present in large quantity in the eye lens. It is made up of acidic (␣A) and basic (␣B) subunits and is also known to be expressed in nonlenticular tissues under stress or disease (1-6). ␣-Crystallin is heatstable (7) and is structurally related to small heat shock proteins (8 -11). Its expression can be induced by thermal (8) or hypertonic stress (12). It interacts with membranes (13) and modulates the intermediate filament assembly (14). Horwitz (15) has shown that ␣-crystallin prevents the aggregation of other crystallins and enzymes. This chaperone-like activity of ␣-crystallin has been further studied in detail by other workers (16 -23). We have earlier investigated the effect of ␣-crystallin on the photoaggregation of ␥-crystallin (19), thermal aggregation of -crystallin, dithiothreitol-induced aggregation of insulin (20), and the rapid refolding of ␤-and ␥-crystallins (21). On the basis of these studies, we postulated that ␣-crystallin prevents aggregation of other proteins by providing appropriately placed hydrophobic surfaces, a structural perturbation above 30°C enhances the chaperone-like activity of ␣-crystallin severalfold.␣-Crystallin from the old human lenses (24) and from the selenite-induced cataractous lenses of an animal model (25) are found to exhibit decreased chaperone-like activity. Our earlier study on refolding of crystallins at high concentrations (21) shows that ␤-or ␥-crystallins aggregate upon refolding while ␣-crystallin does not aggregate. Co-refolding of ␤-crystallin or ␥-crystallin with ␣-crystallin prevents the aggregation under similar conditions. All these studies provide further evidences that the chaperone-like activity of ␣-crystallin is important in the formation and maintenance of the transparency of the eye lens.In the present study, we have observed that human carbonic anhydrase aggregates at 60°C while the aggregation of bovine carbonic anhydrase is negligible at the same temperature. Removal of the...

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Section: ␣-Crystallin Binds To Molten-globulesupporting

confidence: 93%

Section: ␣-Crystallin Binds To Molten-globulementioning

confidence: 99%

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Krishnan

Raman

Rao

1996

Journal of Biological Chemistry

103

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“…710(and references therein), [711][712][713][714][715] BCA II forms a molten globule during refolding both in equilibrium denaturation 678 and transiently during refolding ( Figure 32 parts A and B). 665 716 All four of the characteristics of a molten globule were observed in the refolding of CA.…”

Section: Molten Globulementioning

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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“…This is in contrast to that seen for colicin A, which shows a complete loss of near-UV ellipticity and spectral characteristics at pH 2. Cytochrome c (Ohgushi & Wada, 1983), human carbonic anhydrase B (Jagannadham & Balasubramanian, 1985), and a-lactalbumin (Dolgikh et al, 1981) are other examples of proteins that experience a nearly complete loss of the near-UV spectrum at pH 2. This implies that, in the case of the colicin El P190 polypeptide, a large degree of the tertiary structure is maintained, the environment and mobility of the tryptophan residues is not greatly altered at pH 3, and these tryptophan residues remain significantly restrained at pH 2.…”

Section: Near-u V Specfra Of Pi90mentioning

confidence: 99%

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase

Schendel

1994

Protein Science

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The transition of the colicin El channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid p H values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state.The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at p H 3-6 up to 70 "C implies that the colicin E l channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 "C in the amplitude of the near-UV C D is consistent with preservation of part of the hydrophobic core in a destabilized state at p H 2. Thus, the unfolded state associated with colicin activity at acidic p H has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."The small effect on local membrane acidity of a physiological acidic membrane lipid content, the retention of significant near-UV CD amplitude down to pH 2, and the small extent of immersion of the 40-A globular colicin channel polypeptide in the 10-A lower pH layer at the membrane surface make it unlikely that a local lower pH at the membrane surface significantly facilitates formation of an unfolded intermediate.

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The molten globular intermediate form in the folding pathway of human carbonic anhydrase B

Cited by 54 publications

References 19 publications

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

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

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase

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