Role of disulfide bonds in goose-type lysozyme

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.     

Effect of disulfide bonds on lysozyme dynamics

The effect of destruction of disulfide bonds on the dynamics of proteins was studied by an example of lysozyme by the methods of molecular dynamics. In lysozyme, in the absence of disulfide bonds, the characteristic times of motions of secondary structure devices increased 3-7 times, whereas the amplitudes of fluctuations of secondary structure devices practically did not vary. In the absence of S-S-bonds, the volume of the molecule decreased approximately by 2%, primarily due to a "cleft" between the major and the small domains of lysozyme. Thus, disulfide bonds not only "glue" the secondary structure devices of the protein but also play a role of "rods", maintaining a certain free volume of the molecule necessary for the realization of its functions.     

Evidence for interaction of substrate analogs with chicken eggwhite lysozyme after exhaustive reduction of disulfide bonds

In a study of factors that influence the remaining secondary structure of reduced chicken eggwhite lysozyme, N-acetyl-D-glucosamine (NAG) and N,N'-diacetylchitobiose (di-NAG) were found to alter the circular dichroic (CD) spectrum of the reduced protein and its carboxymethyl derivative (Cml). Thus, negative ellipticities in the far u.v. were greater in the presence of the analogs, with NAG being the more effective. For Cml, curve fitting analysis of the CD data indicated an increased helical content in the presence of NAG by an average of 3% of the chain length, while beta-structure decreased by an equivalent amount. Other compounds structurally related to NAG produced no similar effects on the CD spectrum of Cml, nor were comparable effects of NAG in evidence on the Cm reduced derivatives of ribonuclease, chymotrypsin, wheat germ agglutinin, or alpha-lactalbumin. The effect therefore appears specific between NAG and Cml. Conversion of the tryptophan residue at Position 62 of Cml to the oxindolealanyl derivative prevented these effects of NAG, and this residue may therefore participate in the interaction. During a 4-day incubation at room temperature, the analog preserved the CD spectrum of Cml as well as its concentration. This effect was nearly specific when compared with other Cm reduced proteins and with other carbohydrates. Only one, N-acetyl mannosamine, was effective in preserving the concentration of Cml, but not the CD spectrum. Since D-glucosamine was entirely without effect on either the CD spectrum of Cml or on its change during incubation, the acetyl group appears essential for the NAG-Cml interaction. The specificity between NAG and Cml is tentatively accounted for in terms of interactions with the primary structure, rather than with the remaining secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme reduction of disulfide bonds by thioredoxin. The reactivity of disulfide bonds in human choriogonadotropin and its subunits.

The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, alpha and beta, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12 muM thioredoxin and 0.1 muM thioredoxin reductase at pH 7 all disulfide bonds in the alpha subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the alpha subunit with thioredoxin followed by S-carboxymethylation with iodol[2-3H]acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the alpha subunit were surface oriented and equally reactive. The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.     

Role of individual disulfide bonds in hen lysozyme early folding steps

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-alpha-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-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.     

Characterization of refolded hen lysozyme variant lacking two outside disulfide bonds

We characterized a refolded hen lysozyme variant containing only two SS-bonds, C64-C80 and C76-C94 (4CAHEL). From CD spectra and its activity, it was found that the refolded 4CAHEL has a structural topology analogous to wild-type lysozyme (WTHEL). Moreover, the refolded 4CAHEL showed no thermal transition, indicating that it had a character like a molten globule.     

A laser Raman study of lysozyme denaturation

The Raman spectrum of chemically denatured lysozyme was studied. The denaturants studied included dimethyl sulfoxide, LiBr, guanidine · HCl, sodium dodecyl sulfate, and urea. Previous studies have shown that the amide I and amide III regions of the Raman spectrum are sensitive to the nature of the hydrogen bond involving the amide group. The intensity of the amide III band at 1260 cm^?1 (assigned to strongly hydrogen-bonded α-helix structure) relative to the intensity of the amide III band near 1240 cm^?1 (assigned to less strongly hydrogen-bonded groups) is used as a parameter for comparison with other physical parameters used to assess denaturation. The correlation between this Raman parameter and denaturation as evidenced by enzyme activity and viscosity measurements is good, leading to the conclusion that the amide III Raman spectrum is useful for assessing the degree of denaturation. The Raman spectrum clearly depends on the type of denaturant employed, suggesting that there is not one unique denatured state for lysozyme. The data, as interpreted, place constraints on the possible models for lysozyme denaturation. One of these is that the simple two-state model does not seem consistent with the observed Raman spectral changes.     

Selective reduction of the disulfide bonds of ovine placental lactogen

Reduction and carbamidomethylation of two of the three disulfide bridges of ovine placental lactogen was accomplished by the use of 20-fold molar excess of dithiothreitol over protein disulfide content. The derivative retained its binding capacity to somatogenic as well as lactogenic rat liver receptors, although the latter was somewhat diminished. The two disulfide bonds exposed to the reducing agent are those located near the carboxy- and amino-terminus, while the larger loop remained intact after reduction. This behaviour is similar to that of bovine growth hormone, where the larger loop was also more resistant to reduction.     

Role of disulfide bonds in folding and secretion of human lysozyme in Saccharomyces cerevisiae

We examined folding and secretion of human lysozyme using four mutants each lacking two cysteines expressed in a yeast secretion system. Our results have revealed that the formation of the disulfide bond Cys6/Cys128 in human lysozyme is a prerequisite for correct folding in vivo in yeast. Substitution of Ala for Cys77 and Cys95 gave eight-fold greater secretion of a molecule with almost the same specific activity as that of the native enzyme. Substitutions of the other cysteines gave molecules that were secreted at a lower rate and had lower specific activities than the native enzyme. These are the first findings that the individual disulfide bonds of human lysozyme have different functions in folding and secretion in vivo.     

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.     

Superoxide involvement in the reduction of disulfide bonds of wheat gel proteins

A soluble epoxide hydrase which catalyzes the hydration of 9,10-epoxypalmitic acid has been partially purified from cell-free preparations from $\text{Bacillus}\text{megaterium}$ ATCC 14581. The hydrase can be cleanly separated from a soluble cytochrome P-450-dependent monooxygenase complex, previously demonstrated in this bacterium, that can catalyze the epoxidation of palmitoleic acid.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins. Correspondence to: J. M. Sanchez-Ruiz