Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

Denton, Mary E.; Scheraga, Harold A.

doi:10.1007/bf01024786

Cited by 17 publications

(22 citation statements)

References 59 publications

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“…The 'H NMR spectra of lysozyme and CM6'127-lysozyme have been completely assigned at 35 °C and pH 3.8 (Redfield & Dobson, 1988;Radford etal., 1991). Both proteins are native under these conditions with virtually identical structures, as judged by NMR and X-ray crystallography (Radford et al, 1991;Hill et al, 1993) and by optical methods (Denton & Scheraga, 1991); indeed, the three-disulfide derivative largely retains its enzymatic activity (Radford et al, 1991). At this temperature, however, although CM6-127-lysozyme is still folded, the majority of backbone amides exchange, some to a substantial degree, with bulk solvent during the 4 h acquisition time of a COSY experiment, whereas amides in the unmodified protein are stable against exchange for weeks under these conditions (Pedersen et al, 1991).…”

Section: Resultsmentioning

confidence: 98%

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Eyles¹,

Radford²,

Robinson³

et al. 1994

Biochemistry

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Quenched-flow hydrogen exchange labeling, monitored by 1H NMR and electrospray ionization mass spectrometry (ESI-MS), has been employed in conjunction with stopped-flow circular dichroism and fluorescence to study the kinetic refolding from guanidinium chloride of a derivative of hen lysozyme in which one of the four disulfide linkages (Cys6-Cys127) has been selectively chemically reduced and carboxymethylated (CM6,127-lysozyme). Removal of this disulfide bridge has little effect on the structure and activity of the native enzyme, and the overall kinetics of refolding are very similar to those of the unmodified protein. A substantial amount of secondary structure is formed within 2 ms of the initiation of folding, followed by the slower formation of tertiary interactions characteristic of the native state, which are attained with a time constant (tau) of ca. 200 ms. There is clear evidence for fast and slow refolding populations, as in the intact protein. Folding of the three-disulfide derivative does, however, exhibit a major difference from that of the intact protein under the same final refolding conditions, in that the transient intermediate on the major refolding pathway of the intact protein, having persistent structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labeling during folding of the three-disulfide derivative. This suggests that the disulfide bond linking the N- and C-terminal regions of the protein is crucial for stabilization of the partially folded intermediate. In addition, the overshoot in the far-UV CD and the fluorescence minimum, both of which are attributed to non-native interactions, is not observed in the folding of CM6,127-lysozyme. That the lack of a detectable stable intermediate in the folding of CM6,127-lysozyme does not significantly affect the rate of attainment of the native state of the protein supports the proposed independent nature of the two folding domains and, as the Cys6-Cys127 disulfide bond is located in the alpha-domain, indicates that the rate-limiting step in folding of the intact protein, as well as of the three-disulfide derivative, involves stabilization of the beta-domain. The role of disulfide bridges in the formation and maintenance of the three-dimensional fold of proteins and in facilitating the observation of marginally stable intermediate species is discussed.

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Section: Resultsmentioning

confidence: 98%

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Eyles¹,

Radford²,

Robinson³

et al. 1994

Biochemistry

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“…The 6,127-rcm derivative of lysozyme is thermodynamically less stable than unmodified lysozyme (18,19), which might suggest that it is the unfolded protein that is recognized by the Ub-protein ligase, E3a. We have found that this is not the case.…”

Section: Resultsmentioning

confidence: 99%

“…Two-dimensional NMR measurements by Radford et al (18) support our conclusion that the N-terminal portion of 6,127-rcm-lysozyme is virtually the same as that of the native protein. From an immunochemical study, however, it appeared that the 6,127-rcmlysozyme N-terminal region may be distorted relative to the native conformation (19). The immunochemical and structural data can be reconciled if the N-terminal epitopes that were probed also included Cys-6 or distal portions of the antigen such as its C terminus.…”

Section: Resultsmentioning

confidence: 99%

“…A reduced and carboxymethylated derivative, 6,127-rcm-lysozyme, can mimic this three-disulfide degradation intermediate (17). Three-disulfide lysozyme offers a unique opportunity to probe ubiquitination specificity because, unlike other E3a substrates described thus far, it retains a substantially structured, native-like conformation (17)(18)(19). We report here the x-ray crystal structure of 6,127-rcm-lysozyme determined to a resolution of 1.9 A.…”

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confidence: 99%

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Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Hill

Johnston

Cohen

1993

Proc. Natl. Acad. Sci. U.S.A.

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Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3a (ubiquitin-protein ligase) and E2 (ubiquitincarrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3a-E2 complex are separated by -20 A. Intracellular protein degradation is remarkable for its combination of extreme selectivity and the ability to accommodate an enormous variety of substrates. Protein half-lives in vivo span several orders of magnitude. Moreover, mutations, translational errors, mislocalization, and chemical damage all can lead to polypeptides that are rapidly degraded (1-5). How such proteins are distinguished from their long-lived counterparts is largely unknown. In eukaryotes, a major route for intracellular proteolysis involves covalent modification of protein lysine(s) with the protein ubiquitin (Ub) and subsequent degradation by the 26S Ub-dependent protease complex (6-8). Specificity resides, at least in part, with the Ub-protein ligase that marks a substrate for recognition by the protease. This process is best understood for the E3a ligase from rabbit reticulocytes and the related UBRIencoded enzyme from yeast (9-13).One important E3a recognition determinant is the substrate's N terminus, where only a subset of amino acids is permissive for ubiquitination (9-12). A permissive N terminus is not suffi'cient, however, and the relative orientations and distances that separate lysine ubiquitination sites from

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“…Disulfide bonds can make a substantial contribution to protein stability. The effect of disulfide bonds on stability has been studied in many small globular proteins, such as ribonuclease (Pace et al 1988) and lysozyme (Matsumura et al 1989; Denton and Scheraga 1991; Taniyama et al 1992). For antibodies, the effect of a disulfide bond on stability has been investigated using various isolated domains (Goto and Hamaguchi 1979; Glockshuber et al 1992; Proba et al 1997; Thies et al 2002).…”

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confidence: 99%

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody C_H3 domain

et al. 2008

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Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of b-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C H 3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C H 3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C H 3 dimer were similar, but differences were observed. The reduced C H 3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C H 3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C H 3 domain. Hence, these results may have implications for the stability of the intact antibody.Keywords: human antibody; C H 3 domain; disulfide bond; stability; dimerization With the recent advances in heterologous expression of antibodies, more than a dozen monoclonal antibodybased drugs have reached the market. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. A number of key scientific and technological advancements, such as the generation of fully human antibody by transgenic mice (Green 1999), have contributed to the successful development of antibody-based therapeutics. Stability and homogeneity of therapeutic antibodies are of prime importance for safety and efficacy. Undesired biochemical, structural, and conformational forms can lead to loss of efficacy and risk of adverse side effects.IgG1 (hereafter referred to as antibody) is a complex molecule, composed of two identical light (L) and heavy Reprint requests to: Masazumi Matsumura,

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Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

Cited by 17 publications

References 59 publications

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody C_H3 domain

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Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

Cited by 17 publications

References 59 publications

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Kinetic Consequences of the Removal of a Disulfide Bridge on the Folding of Hen Lysozyme

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain

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Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody C_H3 domain