Compact state of a protein molecule with pronounced small-scale mobility: bovine ?-lactalbumin

Долгих, Д. А.; Lv, Abaturov; Ia, Bolotina; Бражников, Е. В.; Ve, Bychkova; Ri, Gil'manshin; YuO, Lebedev; Semisotnov, Gennady V.; Ei, Tiktopulo; Ob, Ptitsyn

doi:10.1007/bf00256531

Cited by 269 publications

(176 citation statements)

References 57 publications

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“…The molten globule state of bovine a-lactalbumin has been most extensively characterized at pH 2 (Dolgikh et al, 1985;Baum et al, 1989;Alexandrescu et al, 1992;Chyan et al, 1993;Shimizu et al, 1993). However, several observations have dissuaded us from studying bovine a-lactalbumin under these conditions.…”

Section: Resultsmentioning

confidence: 99%

“…The present study exploits both techniques to study the molten globule state of a-lactalbumin, one of the best characterised stable folding intermediates (Dolgikh et al, 1981(Dolgikh et al, , 1985Baum et al, 1989;Ewbank and Creighton, 1991 ;Alexandrescu et al, 1992). a-Lactalbumins are known to form molten globule states under a variety of denaturing conditions (extreme pH, removal of bound calcium, reduction of disulphide bonds, high temperature (Kuwajima, 1989).…”

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confidence: 99%

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Probing Protein Structure by Solvent Perturbation of NMR Spectra

Improta

Molinari

Pastore

et al. 1995

European Journal of Biochemistry

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We have characterized the high-temperature molten globule state of bovine a-lactalbumin by a combined use of photochemically induced dynamic nuclear polarization and nitroxide surface perturbation. Both techniques are extremely well suited to follow the progressive increase of exposed surfaces in the native state and the appearance of partially or completely unfolded species. Our results suggest that the molten globule state obtained at high temperature and pH 7, and the state obtained at pH 2 are not only thermodynamically but also structurally very similar.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Probing Protein Structure by Solvent Perturbation of NMR Spectra

Improta

Molinari

Pastore

et al. 1995

European Journal of Biochemistry

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“…Thus, all-or-none transitions occur not only between N and U [14] and Native MG [12,13], but also between MG and essentially U. This suggests that globular proteins can exist in at least three discrete states: N, MG and U.…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown [1,12,13] that the transition between N and MG is tl'te 'all-or-none' one, just as in other types of protein denaturation [14]. However, the question remained as to whether MG is separated from U by another all-or-none transition, or if it is no more than a limiting case of the 'squeezed' coil and which can gradually 'swell' into a lull U.…”

Section: Introductionmentioning

confidence: 99%

‘All‐or‐none’ mechanism of the molten globule unfolding

et al. 1992

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The Gdm-HCl-induced unfolding of bovine carbonic anhydrase B and S. at~ret~s fl-lactamase was studied at 4°C by a variety of methods. With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal. This means that equilibrium intermediates between the molten globule and the unfolded states are about, i.¢. the molten globule unfolding follows the "all-or-none' mechanism.

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“…An alternative possibility might be the disruption by ANS of preexisting aggregates, which could be a first order process. However, although the low pH form of carbonic anhydrase does indeed seem to be associated (Semisotnov et al, 1991), that of bovine a-lactalbumin, which shows qualitatively similar timedependent behavior with ANS, has been shown to be monomeric (Dolgikh et al, 1985). It therefore seems unlikely that changes in the association state of the protein can provide a general explanation of the time-dependent behavior observed.…”

Section: Interaction With Equilibrium Molten Globulesmentioning

confidence: 89%

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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Compact state of a protein molecule with pronounced small-scale mobility: bovine ?-lactalbumin

Cited by 269 publications

References 57 publications

Probing Protein Structure by Solvent Perturbation of NMR Spectra

Probing Protein Structure by Solvent Perturbation of NMR Spectra

‘All‐or‐none’ mechanism of the molten globule unfolding

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

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