Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe

Semisotnov, Gennady V.; Rodionova, N. A.; Razgulyaev, O.I.; Uversky, Vladimir N.; Gripas, A. F.; Gilmanshin, Rudolf

doi:10.1002/bip.360310111

Cited by 1,267 publications

(1,032 citation statements)

References 29 publications

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“…These observations are suggestive of a molten-globule-like character of the I1 state; a similar type of molten-globule state was reported previously for human acidic fibroblast growth factor (hFGF-1) (47,48). The I2 state has substantial secondary structure and also shows weak ANS binding, which suggests that this state is a partly condensed state with a small solvent-accessible non-polar cluster.…”

Section: Discussionsupporting

confidence: 82%

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Shukla

Singh

Vispute

et al. 2017

Biophysical Journal

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Cyclophilin catalyzes the ubiquitous process ''peptidyl-prolyl cis-trans isomerization,'' which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1 H-15 N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

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Section: Discussionsupporting

confidence: 82%

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Shukla

Singh

Vispute

et al. 2017

Biophysical Journal

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“…As discussed above, binding of ANS to the equilibrium molten globule state of bovine carbonic anhydrase B populated at high pH in the presence of intermediate concentrations of guanidinium chloride has been shown to cause an increase in its fluorescence that is complete within the dead time of a stopped-flow experiment (Semisotnov et al, 1991). We obtained the same result under these conditions (data not shown) but found quite different behavior for another partially folded state of this same protein, formed in this case at low pH, as shown in Figure 1.…”

Section: Interaction With Equilibrium Molten Globulesmentioning

confidence: 94%

“…This probe has now become almost standard for diagnosing the population of compact, partially folded states, usually referred to as molten globules (Semisotnov et al, 1991).…”

Section: Engelhard and P A Evansmentioning

confidence: 99%

“…If partially folded states of proteins have clusters of hydrophobic side chains that are not yet fully occluded in the native core structure, these might also provide binding sites for the dye and, indeed, molten globule states of several proteins, generated under mild denaturing conditions and shown by viscometric and scattering measurements to have collapsed structures interact very effectively, leading to large fluorescence intensity increases (Tandon & Horowitz, 1989;Semisotnov et al, 1991).…”

Section: Engelhard and P A Evansmentioning

confidence: 99%

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Kinetics of interaction of partially folded proteins with a hydrophobic dye: Evidence that molten globule character is maximal in early folding intermediates

1995

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Interaction with 8-anilino-1-naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates.Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and a-lactalbumin have been compared with conventional single-mix experiments and large discrepancies observed. When ANS was present throughout refolding, time-dependent changes attributed to the formation or reorganization of protein-ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding.The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states. sitive to side-chain clustering, but they are not necessarily very representative of global structure and the total fluorescence intensity, which is the parameter most easily followed, is difficult to interpret in detail. Combinations of such simple methods with protein engineering offer a more general approach (Matthews, 1993;Serrano, 1994), but here too there are potential problems if the clustering is rather tenuous or nonspecific. It is clear neither whether such behavior would necessarily be detected by simple spectroscopic methods nor how mutations ought to be analyzed in the case of nonspecific interactions. One technique that appears to go some way to addressing the problem uses 8-anilino-1-naphthalene-sulfonate (ANS) as an extrinsic probe. This dye tends to b...

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“…The partially folded intermediates have a wide distribution from a high ordered molten globule (MG) (close to the native state) to a pre-molten globule (close to the unfolded state), with solvent-accessible hydrophobic regions (3,4). By definition, a molten globule state would share a compact globular structure with the native conformation.…”

Section: Introductionmentioning

confidence: 99%

Partially folded intermediates during trypsinogen denaturation

Martins

Santoro

1999

Braz J Med Biol Res

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The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was DG H 2 O = 6.99 ± 1.40 kcal/ mol for guanidine hydrochloride and DG H 2 O = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

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Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe

Cited by 1,267 publications

References 29 publications

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Kinetics of interaction of partially folded proteins with a hydrophobic dye: Evidence that molten globule character is maximal in early folding intermediates

Partially folded intermediates during trypsinogen denaturation

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