NMR Structural Study of Two-Disulfide Variant of Hen Lysozyme:  2SS[6−127, 30−115]A Disulfide Intermediate with a Partly Unfolded Structure

Noda, Yasuo; Yokota, Atsushi; Horii, Daisuke; Tominaga, Takeshi; Tanisaka, Yoshiaki; Tachibana, Hideki; Segawa, Shin-ichi

doi:10.1021/bi0113418

Cited by 19 publications

(39 citation statements)

References 22 publications

(44 reference statements)

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“…[10,37] Of the 129 residues, 107 could be assigned for 2SS a in water (pH 3.8); resonance assignments were mainly missing for residues in the b-domain. The assignments obtained mostly agreed with those of Noda et al [30] The 1 H, 15 N-HSQC spectrum of partly folded 2SS a in water (pH 3.8) contains more than the expected 127 backbone NH resonances. Some of the unassigned peaks were considerably weaker than the other resonances.…”

Section: Resultssupporting

confidence: 85%

“…Other proteins fail to fold in the absence of some disulfide bridges; for example, the absence of two of the disulfide bridges in the b-domain of hen-egg-white lysozyme (between cysteines 64-80 and 76-94) destroys its ability to refold. [29,30] We have studied hen-egg-white lysozyme mutants in which the disulfide bonds have been removed. Lysozyme contains two structural domains: the a-domain, which contains residues 1-35 and 85-129, and the b-domain, which comprises residues 36-84.…”

Section: Introductionmentioning

confidence: 99%

“…This group was able to show that the protein is partially folded in water at pH 3.8 whereas the 2SS b variant, which has intact disulfide bridges in the bdomain only, was unfolded under the same conditions. [29,30,36] …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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This report describes NMR-spectroscopic investigations of the conformational dynamics of disulfide bonds in hen-egg-white lysozyme substitution mutants. The following four systems have been investigated: 2SS(alpha), a lysozyme variant that contains C64A, C76A, C80A and C94A substitutions, was studied in water at pH 2 and 3.8 and in urea (8 M, pH 2); 2SS(beta) lysozyme, which has C6S, C30A, C115A and C127A substitutions, was studied in water (pH 2) and urea (8 M, pH 2). The NMR analysis of heteronuclear 15N-relaxation rates shows that the barrier to disulfide-bond isomerisation can vary substantially in different lysozyme mutants and depends on the residual structure present in these states. The investigations reveal cooperativity in the modulation of micro- to millisecond dynamics that is due to the presence of multiple disulfide bridges in lysozyme. Mutation of cysteines in one of the two structural domains substantially diminishes the barrier to rotational isomerisation in the other domain. However, the interactions between hydrophobic clusters within and across the domains remains intact.

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Section: Resultssupporting

confidence: 85%

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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“…The data obtained herein indicate that once folded, the two TCI domains display only marginal differences in their conformational stability. In addition, their disulfide bonds are similarly protected: Cys 3 , Cys 16 , Cys 31 , and Cys 32 in the N-terminal domain, together with Cys 40 , Cys 64 , Cys 70 , and Cys 71 in the C-terminal one expose less than 10% of their surface to solvent. This locking in of disulfides explains why both intermediates can be largely and simultaneously detected during the oxidative folding and reductive unfolding of TCI, giving rise to symmetric folding and unfolding routes as schematically shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

Arolas

Pantoja-Uceda

Ventura

et al. 2008

Journal of Biological Chemistry

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There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N-and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.Understanding the sequence of folding events that lead to a biologically active protein from its amino acid sequence is one of the major challenges in structural and molecular biology. The initial theoretical efforts devoted to the study of protein folding have been reinforced in the last several years by the discovery of a wide range of pathological diseases associated with protein misfolding and the increasing pharmacological interest in protein drug design (1, 2). The structural characterization of partially folded intermediates and the analysis of the interactions that stabilize them are of fundamental importance to unveil the folding mechanisms (3, 4). But these studies are hampered by the rapid and cooperative nature of protein folding, and therefore, the short half-life of the intermediates arising during the folding reaction. It is however possible to trap and isolate discrete intermediates from the oxidative folding of disulfide-rich proteins because of the particular chemistry of disulfide bond formation that takes place as an integral part of disulfide folding (5, 6).A large set of single domain, disulfide-rich proteins has been investigated to date in terms of oxidative folding; among them, outstanding examples such as bovine pancreatic trypsin inhibitor (BPTI), 5 ribonuclease A (RNase A), hirudin, insulin-like growth factor-1 (IGF-1), or conotoxins (7-11). These folding studies have involved the trapping (either by acidification or alkylation), isolat...

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“…Titrations were carried out by adding microliter aliquots of aqueous Cu-cyclam (125 mM, pH 4.6). The assignments for 1 H NMR resonances of HEWL are based on those given by Noda et al (22).…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Protein recognition of macrocycles: Binding of anti-HIV metallocyclams to lysozyme

Hunter

McNae²,

Liang³

et al. 2005

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

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(Fig. 1) and is a highly potent anti-HIV drug (2, 3). AMD3100 has recently been on clinical trial for the treatment of AIDS. Additional medical interest in cyclams arises from their ability to mobilize stem cells and to carry diagnostic and therapeutic radionucleotides (4), and from their potential use in transplant, inflammation, and cancer therapy (5). Metal ions such as Zn 2ϩ and Cu 2ϩ bind to cyclam strongly (log K values Ϸ15 and 27, respectively (6)) and relatively rapidly (7), and it seems likely that metal complexation by xylyl-bicyclam is involved in the mechanism of action of the drug in vivo. For metal-bicyclam complexes, there is a close correlation (8) between antiviral activity and binding to the coreceptor CXCR4 as monitored by inhibition of 12G5 mAb binding and the intracellular Ca 2ϩ signal induced by SDF-1␣ chemokine, the order of decreasing activity being Zn 2ϩ Ϸ Ni 2ϩ Ͼ Cu 2ϩ Ͼ Ͼ Co 3ϩ Ͼ Ͼ Pd 2ϩ . The affinity of AMD3100 for the CXCR4 receptor is enhanced by factors of 7, 36, and 50 by incorporation of Cu 2ϩ , Zn 2ϩ , or Ni 2ϩ , respectively, into the cyclam rings (9), and a similar metal-induced enhancement in CXCR4 affinity is observed for cyclam itself, although the affinity for the receptor is much less. Aspartate carboxylate groups of CXCR4 (especially Asp-171 and Asp 262) have been implicated in the bicyclam binding site (9, 10), and models of CXCR4 containing (carboxylate)O⅐⅐⅐HN(cyclam) H bonding and carboxylate(O)-metal(cyclam) coordination have been built (9-11).Metal cyclam complexes have a potentially rich configurational chemistry (4, 12). Each of the coordinated N atoms is chiral. The metal ion can commonly be 4-, 5-, or 6-coordinate, and symmetrical trans configurations can fold to give cis structures, as illustrated for trans-V in Fig. 1. In view of the targeting of anti-HIV cyclam derivatives to CXCR4, it is important to understand the nature of the interactions that determine their binding sites on proteins and in particular whether proteins can recognize different configurations of metal cyclams selectively. However, there appear to be no reported structures of protein

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NMR Structural Study of Two-Disulfide Variant of Hen Lysozyme: 2SS[6−127, 30−115]A Disulfide Intermediate with a Partly Unfolded Structure

Cited by 19 publications

References 22 publications

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

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

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

Protein recognition of macrocycles: Binding of anti-HIV metallocyclams to lysozyme

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