To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-␣-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-␤-domain (C64-C80) and the inter-␣␤-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.Keywords: Lysozyme; disulfide; mutants; folding kinetics; secondary structure Much of our understanding of the basic principles that govern protein folding has been acquired by studying the renaturation of small proteins in vitro. Among these, hen lysozyme has been the most extensively investigated by the use of a wide array of fast-kinetics methods based on a variety of spectroscopic and chemical signals. These include absorption, intrinsic fluorescence of the aromatic residues, dynamic fluorescence quenching, fluorescence of extrinsic probes such as 8-anilino-1-naphthalenesulfonic acid (ANS) or the substrate analog 4-methylumbelliferyl-N,NЈ-diacetyl-␤-chitobiose, circular dichroism, and pulsed proton exchange followed by nuclear magnetic resonance (NMR) or mass spectroscopy . These studies, however, failed to reflect the folding of the newly synthesized polypeptide chain, because they deal with a protein in which the four disulfide bonds of the native molecule were kept intact in the denatured state; thus, they were already present at the onset of the renaturation. This contrasts with the physiological folding process, which starts with the reduced protein. This is of importance in view of the marked difference in the rates and efficiencies of renaturation of Abbreviations: ANS, 8-anilino-1-naphthalene-sulfonic acid; CD, circular dichroism; GuHCl, guanid...

We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 M oxidized glutathione, and 20 M reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi-or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.Since Anfinsen's work on the in vitro renaturation of unfolded ribonuclease A (1), it is commonly accepted that all the information required for a protein to fold properly is contained in its amino acid sequence. However, the code that allows the formation of a fully folded protein from its amino acid sequence has not yet been deciphered. Three models are currently proposed to describe this process. The framework model is a sequential model in which secondary structure elements form first followed by a tighter packing of the molecule (2). Another model assumes that the polypeptide chain undergoes a rapid collapse driven by hydrophobic forces that would yield an intermediate close to the molten globule (3). In the puzzle model (4) structural elements form at different sites on the polypeptide chain, and their formation induces further folding of the whole protein.According to experimental data, it is not yet possible to determine which model most accurately describes the folding mechanism. However, some general features of protein folding have arisen. Stopped-flow circular dichroism studies showed that a large amount of secondary structure is formed in the dead time (milliseconds time range) of the observation. This has been observed for proteins such as ␣-lactalbumin and lysozyme (5), dihydrofolate reductase (6), and holocytochrome c (7). On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8, 9). Using both techniques to observe the folding of the same protein, as was the case for cytochrome c (7), lysozyme (10, 11), and interleukin-1␤ (12), secondary structures coul...

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism~CD!, fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and0or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.

The authors have studied the temperature effect on the luminescence of spinach chloroplast suspension after illumination at 675 nm.The decay of the luminescence has been followed at constant temperature (from 0°C to 40°C) or after sudden temperature jumps (0°C 5 AT s 47°C).They have compared the results obtained with intact chloroplasts and those after mild thermal denaturation.From these experimentally found values they have calculated the activation energies related to the radiative and non-radiative ways of disappearence of the entity which is at the origin of the luminescence.On the basis of the values found for these energies (0.9 eV and 0.43 eV respectively), the authors evolve the hypothesis that this entity may be an electron self-trapped in the chloroplast structure (small polaron). I N T R O D U C T I O N STREHLER et Arnold ont montre en 195 1 que des vegetaux chlorophylliens-microorganismes et plantes superieures [ 13 -ainsi que les chloroplastes isoles [2] -emettaient de la lumikre pendant plusieurs minutes aprks la cessation de leur Cclairement. Cette luminescence prolongCe apparait comme une propriCte trks gCnCrale des organismes photosynthitiques [3]. Ses caracteristiques ont Ct C precisees par de nombreux auteurs: son spectre d'emission est celui de la chlorophylle excitCe in vivo [4,5]; son intensite depend de celle de la lumikre excitatrice [23 et de la temperature: elle atteint son maximum a environ 37°C pour les chlorelles[l] et 36°C pour les chloroplastes[2]. Un chauffage de 4 min B 51°C Climine 95% de la luminescence des chlorelles[6] et 5 min de chauffage ii 65°C entrainent la disparition quasi-totale (99%) et irrkversible de I'emission lumineuse des chloroplastes [71.La cinetique de la dkcroissance de cette luminescence aprks l'kclairement a ete Ctudi6e dans des domaines de temps trks varies allant de la zone de la milliseconde a celle de l'heure. Elle a Ct C diversement analysee B l'aide de fonctions de la forme: t -I et t --2 ou par des decompositions en plusieurs composantes: 2 composantes dont les demi-vies sont respectivement de 0.01 sec et de 1 sec [3], 5 composantes exponentielles dont les durees de vie s'echelonnent de 5 X B 15 sec pour des temperatures allant de moins 196" plus 40°C [8,9].Une fraction de I'energie lumineuse absorbee par les plantes est donc emmagasinke dans des conditions telles qu'elle puisse Gtre partiellement restituee sous forme de luminescence. Cependant le rendement quantique de cette emission parait faible: Tollin, Fujimori et Calvin ont trouve, aprks un eclair, des valeurs de I'ordre de 10-g [5].Si faible que soit la part de cette luminescence dans le bilan CnergCtique des chloro-I39