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Molten-Globule’ State Accumulates in Carbonic Anhydrase Folding

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Molten-Globule’ State Accumulates in Carbonic Anhydrase Folding View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 165, number 1 FEBS 1079 January 1984 ‘Molten-globule’ state accumulates in carbonic anhydrase folding D.A. Dolgikh, A.P. Kolomiets, I.A. Bolotina* and O.B. Ptitsyn+ Institute of Protein Research, USSR Academy of Sciences, 142292 Pushchino, Moscow Region and *Institute of Molecular Biology, USSR Academy of Sciences, II 7312 Moscow, USSR Received 17 October 1983 Kinetics of folding and unfolding of bovine carbonic anhydrase B were monitored by circular dichroism, viscometry and esterase activity. It was shown that kinetic intermediate states accumulating in folding process reveal a native-like compactness and secondary structure but have a symmetrized average environment of aromatic side groups and no esterase activity. These properties allow one to consider these intermediate states as the ‘molten-globule’ state of a protein molecule previously described by us for several equilibrium forms of bovine and human Lu-lactalbumins and bovine carbonic anhydrase B. Carbonic anhydrase B Protein folding Molten-globule state of protein molecules Folding intermediate 1, INTRODUCTION this model the increase of fluctuations is due to a slight increase of the molecular volume leading to Recently [l-3] we have described a new equilib- a sharp decrease of Van der Waals intramolecular rium state of a protein molecule, ‘compact globular interactions [4]. state with fluctuating tertiary structure’, using the Quite recently we have found that a similar in- examples of bovine and human a-lactalbumins. termediate state also exists in bovine carbonic an- Both proteins can be transformed into this state hydrase B at pH -3.6 [3,5]. CAB at pH 3.6 has a under the influence of various agents: acid pH, compact structure with a far UV CD spectrum even moderate concentrations of Gu-HCI, heating, etc. more pronounced than in the native state but with A protein molecule in this state is nearly as com- a near UV CD spectrum similar to the completely pact as native protein and has a native-like secon- unfolded state [6]. We have shown [S] that CAB dary structure, but at the same time it has a sym- at pH -3.6 has a native-like secondary structure metrical average environment of aromatic and (evidenced from its infrared spectrum) and that the other side groups, no cooperative temperature main part of its molecule has fast deuterium ex- melting and fast deuterium exchange. A model of change and does not melt cooperatively on heating. this ‘intermediate’ (between the native and un- Therefore all properties of the main part of its folded states) state has been proposed [l-4] as a molecule at pH -3.6 correspond to the properties compact globule with pronounced fluctuations of of the intermediate state described earlier [l-3] for its three-dimensional structure (first of all with cu-lactalbumins. It has also been shown that CA B large fluctuations of its side groups). According to has a similar state at -2 M Gu-HCl as it has far and near UV spectra similar to those at pH 3.6 [7,8], it is compact [9] and does not melt coopera- + To whom correspondence should be addressed tively on heating (unpublished). Abbreviations: CA B, carbonic anhydrase B; a-LA, LY- A similar intermediate state has also been found lactalbumin; Gu-HCI, guanidine hydrochloride; CD, for ribonuclease A [lo] and for cytochrome c [ 111. circular dichroism; UV, ultraviolet; states of CA B - N Wada proposed for this state the term ‘molten (native), I (intermediate) and U (unfolded) globule’ [ 111. The existence of a similar inter- Published by Elsevier Science Publishers B. V. 88 00145793/84/$3.00 0 1984 Federation of European Biochemical Societies Volume 165, number 1 FEBS LETTERS January 1984 mediate or molten globule state for such different refractive index at 589 nm [20]. proteins as a-lactalbumins, carbonic anhydrase, Measurements of transitions from the inter- ribonuclease and cytochrome c suggests that this mediate state (at 1.97 M Gu-HCl) to the other state is not a rare exception and may be of general states are hampered by the slow (half-time -2 h) importance for protein physics. transitions of CAB at -2 M Gu-HCl to an irre- Carbonic anhydrase folds much slower than a- versibly denatured state [9]. Although in our ex- lactalbumins, ribonuclease and cytochrome c (see periments this effect was substantially smaller than e.g. [12]) which facilitates the investigation of the in [9], to exclude it completely we transferred CA B possible role of this intermediate state in its folding to 1.97 M Gu-HCI from 5.45 M Gu-HCI 5-10 min pathway. The kinetics of CAB refolding and before the transition from 1.97 M Gu-HCl to the unfolding have been studied by several authors other states. [6,9,13-181. Complex (multi-phase) kinetics of Time courses of molar ellipticities [8]222 and both processes have been shown [6,15-181 which, [8]270have been monitored by dichrograph (Marks taken by itself, does not prove the existence of III-S, Jobin Ivon) directly during the transition of kinetic intermediates as CAB has 20 prolines and CAB from the initial to final state in the spectro- therefore may have several unfolded forms dif- photometric cell. The protein concentration in CD fering in proline isomerization [ 121. However it has measurements was usually -1 mg/ml and the path been shown [17] that the secondary structure of length of the cell was 1 cm for the near UV and CA B (monitored by the far UV CD spectra) is re- 0.5 mm for the far UV experiments. stored in the course of folding much faster than the Specific viscosity p,, was measured with a capil- tertiary one (monitored by [8]270) which is good lary viscometer (Viscomatic) with an automated evidence for the existence of intermediate(s) with solution pump which permits monitoring of the secondary structure but without a specific tertiary time course of vsp for the same protein sample by one. It has also been shown [9] that the esterase the change of its flow time through the capillary. activity of CA B is restored slowly in the course of The viscometer was thermostated to within 0.01 “C. protein folding and that the rates of this recovery CA B concentration in viscosity measurements was are similar at the transitions both from the un- -3 mg/ml, the duration of each measurement folded state (5 M Gu-HCl) and from the inter- being -100 s. mediate one (2 M Gu-HCl). Esterase activity was determined as in [ 191 by the We show here that the state which accumulates increase of absorbance at 348 nm due to hydrolysis during carbonic anhydrase folding has not only a of p-nitrophenylacetate (p-NPA) corrected for the native-like secondary structure but also a native- spontaneous hydrolysis in the absence of the en- like compactness, i.e. it is similar to the inter- zyme. Aliquots (0.1 ml) of the renaturing protein mediate state which exists in this protein at pH 3.6 (-1 mg/ml) were placed into the cell containing and at -2 M Gu-HCl. We also compare the kine- 2 ml of the reactive mixture with 1 mM p-NPA. tics of all 6 transitions between 3 different states of The duration of each measurement was -2 min. As carbonic anhydrase (native, intermediate and un- Gu-HCl is an inhibitor of CA B the activity of the folded). renaturing protein at 0.97 M Gu-HCl was com- pared with the activity of native CAB under the same conditions. 2. MATERIALS AND METHODS The temperature was 19-20°C for all experi- Bovine CA B was obtained from Serva Feinbio- ments. All kinetics were studied at neutral pH chemica and purified on a column with cellulose values. DE-52 (Whatman) as in [19] with small modifica- tions. The homogeneity of the proteins was checked 3. RESULTS by disc-electrophoresis in polyacrylamide gels with and without SDS. 3.1. Kinetics of renaturation Protein concentrations were determined using 3.1.1. U+N transition extinction coefficients E!@&280nm = 18.3 [6]. Gu- Refolding kinetics were studied by diluting CA B HCl concentrations were determined from the solution in 5.45 M Gu-HCI (where molecules are 89 Volume 165, number 1 FEBS LETTERS January 1984 completely unfolded [6]) to 0.97 M Gu-HCl (where 3.1.2. I -+ N transitions the protein is in the native state [6]). Fig. 1 presents Fig.2 represents the refolding kinetics at the the data plotted as the ‘fraction of nativity’ I + N transition of CA B from the molten-glubule state (at 1.97 M Gu-HCl) to the native one moni- fN = (X- XD)/(~N-XD) tored by the parameters which change during this transition ([0]270 and esterase activity). The kinetics where X is the value for the studied parameter at of the U + N transition (monitored by the same a given time and XN and Xn are the values for the parameters) are reproduced from fig.1 for com- native and denatured states, respectively. parison. One can see that kinetics of I + N and Fig.1 shows that the values of reduced viscosity U -+ N transitions are very similar having a half- (reflecting the compactness of molecules) as well as time -25-30 min. the values of [8]222 (reflecting their secondary structure as it has been demonstrated [17] that 3.2. Kinetics of unfolding these values represent well the changes of the whole 3.2.1.
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