Role of individual disulfide bonds in hen lysozyme early folding steps

Abstract

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...

Contributions of Disulfide Bonds in a Nested Pattern to the Structure, Stability, and Biological Functions of Endostatin

“…In the absence of one or all of the disulfide bonds, many proteins cannot sustain such properties (11,13). Each disulfide bond has different roles in the stability and activity of proteins (13)(14)(15)(16)(17). In order to elucidate the functions of the two disulfide bonds in ES, each of the two individual disulfide bonds was mutated by substituting cysteine residues with alanine, which is a conservative change (18).…”

Characterisation of Disulfide‐Bond Dynamics in Non‐Native States of Lysozyme and Its Disulfide Deletion Mutants by NMR

“…Oxidative folding has been studied in great detail in vitro, for example, for ribonuclease A, [16][17][18][19][20] BPTI [21][22][23] and henegg-white lysozyme. [24][25][26][27] Initial in vivo investigations into the process have also been performed, for example, on the HIV-1 envelope glycoprotein. [28] However, many aspects of the role of disulfide bridges in protein folding remain poorly understood.…”

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

“…The average value of the half-life is 11 h. The intermediate was found to be unexpectedly stable when compared with typical folding intermediates with much shorter half-lives on the microsecond-to-millisecond timescale. 3,[5][6][7][8][9][10][11][12][13] These results indicate that the transition from the intermediate to the native state is highly cooperative and requires gross structural rearrangement. (Table 1), compared to typical folding intermediates.…”

“…(Table 1), compared to typical folding intermediates. 3,[5][6][7][8][9][10][11][12][13] In many cases, slow folding reactions are caused by cis/ trans isomerisation of proline residues; that is, for proteins containing cis-proline in its native state, the trans → cis isomerisation slows the folding down because the trans isomer is more favoured in the unfolded state. [29][30][31] However, in the case of SBD, all five proline residues adopt the trans conformation in the native state.…”

“…However, it is difficult to experimentally determine the structural details of these intermediates because of their instability, small population size, and transient nature. 3,[5][6][7][8][9][10][11][12][13] In some cases, it has been indicated that appropriate packing in the hydrophobic core of proteins is critical for efficient folding; insufficient burial and packing of core residues lead to the accumulation of folding intermediates. [10][11][12][13][14][15][16][17] In other cases, it has been shown that interactions formed on protein surfaces are important for efficient folding.…”