Dynamic filtering by two-dimensional 1H NMR with application to phage lambda repressor.

Weiss, Michael A.; Eliason, James L.; States, David J.

doi:10.1073/pnas.81.19.6019

Cited by 38 publications

(20 citation statements)

References 43 publications

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“…The spectrum of the reduced peptide (Fig. 1A) contains a large number of intense intense crosspeaks observed in the spectrum are from residues with substantial independent mobility; such mobility has been observed in a number of other proteins of high molecular weight (22)(23)(24)(25). In the present case the mobile regions are likely to derive from the relatively disordered N and C termini of the Fab polypeptide chain.…”

Section: Resultssupporting

confidence: 56%

Antigen mobility in the combining site of an anti-peptide antibody.

Cheetham

Raleigh

Griest

et al. 1991

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

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The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 5744), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.One of the most intriguing aspects of antibody recognition is the ability of some antibodies raised against peptide fragments of proteins to crossreact with intact native proteins (1-3). This phenomenon not only has considerable significance from the point of view of protein structure and folding but also has generated much interest in the possibility ofusing small peptides in a pharmacological role, particularly as synthetic vaccines (4,5). Little is known, however, about the underlying principles that determine whether a particular peptide is able to act as a good mimic of a protein epitope, primarily-because of the shortage of structural information pertaining to antibodies complexed with their peptide antigens. To our knowledge there has, for example, been only one report ofthe crystallographic structure of an anti-peptide antibody-peptide complex (6). In contrast a number of structures of anti-protein antibodies complexed with their protein antigens have been determined (7-11). As part of a long-term study in our laboratories of the structural origins of antibody recognition and protein-peptide crossreactivity, a panel of antibodies (Gloopl-GloopS) specific for the loop determinant of hen egg lysozyme was raised against a peptide immunogen that incorporated residues 57-84 of the protein sequence (12)-known as the "loop" peptide. This sequence corresponds to a large surface loop in the native protein structure and contains a disulfide bridge between residues 64 and 80. The NMR studies have focused on one of the antibodies, Gloopl. The antibody binds two forms of the loop peptide, where the disulfide is either reduced or oxidized, with equal affinity (Kd 10-8 M). The antibody is also strongly crossreactive with the native protein; it binds to hen lysozyme itself with only a 10-fold drop in affinity (12, 13). Crystallographic studies have yielded a structure for the Gloopl Fab (R.E.G. a...

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Section: Resultssupporting

confidence: 56%

Antigen mobility in the combining site of an anti-peptide antibody.

Cheetham

Raleigh

Griest

et al. 1991

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

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“…Unlike R1_236 and R2-236, R4-236 does not retain operator DNA on a nitrocellulose filter, so its activity was not determined. NMR spectroscopy was performed as described (25). DNA sequencing was performed by the method of Maxam and Gilbert (20 (30) and one from pKB252 (31).…”

Section: Methodsmentioning

confidence: 99%

“…The DNA encoding amino acids 7-236 of repressor was next ligated to an expression vector. ptacl2B (25) contains the strong tac promoter (17,32) and the lac Shine-Dalgarno (SD) sequence, followed by an Nco I site. A fragment of ptacl2B was prepared as the pTR182 fragment above.…”

Section: Methodsmentioning

confidence: 99%

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Eliason¹,

Weiss²,

Ptashne³

1985

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

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Several lines of evidence indicate that the phage X repressor recognizes its operator by using, in part, an a helix (the "recognition helix"), which it inserts into the major groove of DNA. In addition to its recognition helix, X repressor has an "arm," consisting of the first six amino acids, that wraps around the DNA helix. We constructed plasmids that, in Escherichia coli, direct the expression of derivatives of A repressor that lack the NH2-terminal one, three, six, or seven amino acids. We studied these modified proteins in vivo and in vitro, and from our results we argue that the arm: (i) contributes a large portion of the binding energy; (ii) helps to determine sequence specificity of binding and, in particular, the relative affinities for two wild-type binding sites; (iii) determines entirely repressor's response to one operator mutation (a "back-side" mutation); (iv) magnifies repressor's response to other operator mutations ("front-side" mutations); and (v) increases the sensitivity of repressor binding to salt concentration and temperature.Models based upon x-ray crystallographic studies (1-3) and upon sequence homologies (4,5) suggest that many prokaryotic regulatory proteins recognize their binding sites by a common mechanism. According to these models (6-8), such proteins make sequence-specific contacts by inserting an a helix, the "recognition helix," into the major groove of DNA. In one case it has been shown that replacing the recognition helix from one protein with the recognition helix of another alters the binding specificity (9). Phage X repressor is unusual because, in addition to a recognition helix, it also bears a flexible NH2-terminal "arm" that consists of the first six amino acids and evidently wraps around the DNA (10). According to the current picture (6), each monomer of a repressor dimer inserts a recognition helix into the major groove on one face of the DNA helix, the "front side." The two arms of the dimer wrap around the DNA, making specific contacts in the major groove on the opposite face, the "back side," and also making nonspecific contacts to the DNA phosphates. Phage X Cro protein, which lacks an arm, binds to the same operator sites as does repressor. Cro also binds as a dimer, inserting a recognition helix from each monomer into the major groove on the front side of the DNA helix, but does not contact the back side of the operator (7).Previous experiments have shown that phage X repressor derivatives with defects in the arm have a reduced affinity for operator. A point mutation changing Lys-4 to Gln-4 greatly reduced repressor binding (11). Proteolytic removal of the NH2-terminal three amino acid residues decreased the affinity of a repressor fragment [the NH2-terminal domain (12)] for an operator site and altered the ability of the same repressor fragment to protect guanines in the operator from methylation by dimethyl sulfate (10). Since removal of the first three amino acids does not change the global conformation of the protein (13), alterations made to the...

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“…Rather, whereas amide line widths are anomalously broadened in DKP-insulin, those of 3G-DKP-insulin are more uniformly appropriate given the rotational correlation time of a small globular protein. Hence, DQF-COSY H N -H ␣ cross-peaks in the B9 -B19 and A13-A19 helices, essentially unobservable in DKP-insulin because of antiphase cancellation (37), are largely present in the spectrum of 3G-DKP-insulin. Similar trends in amide line widths were observed in comparative studies of insulin and a fragment lacking residues B26 -B30 (des-pentapeptide[B26 -B30]insulin (DPI); Refs.…”

Section: Column 6; See Above)mentioning

confidence: 98%

Mechanism of Insulin Chain Combination

Hua

Chen

Jia

et al. 2002

Journal of Biological Chemistry

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The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal ␣-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the Nterminal ␣-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity.1 H NMR studies of a representative analog lacking invariant side chains Ile A2 and Val A3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise nativelike structure. That this and related partial folds retain efficient disulfide pairing suggests that the native Nterminal ␣-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.

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Dynamic filtering by two-dimensional 1H NMR with application to phage lambda repressor.

Cited by 38 publications

References 43 publications

Antigen mobility in the combining site of an anti-peptide antibody.

Antigen mobility in the combining site of an anti-peptide antibody.

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Mechanism of Insulin Chain Combination

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