Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

Jones, Warren C. Jun.; Rothgeb, T. Michael; Gurd, Frank R. N.

doi:10.1016/s0021-9258(17)32870-3

Cited by 96 publications

(40 citation statements)

References 66 publications

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“…Since fragment 132-153 differs from native myoglobin in size as well as conformation, we used S-methylmyoglobin to see which of these alterations was responsible for the difference in requirements for presentation. S-Methylmyoglobin is similar in size to the native molecule but has been partially denatured and unfolded by the addition of a second methyl group to hydrophobic methionine residues at positions 55 and 131, thereby introducing positive charges into the hydrophobic core of the molecule (20,21). These require the molecule to unfold to admit solvent.…”

Section: Resultsmentioning

confidence: 99%

“…These require the molecule to unfold to admit solvent. NMR studies of [13C]methyl groups introduced into the methionine residues, showing narrow resonances with equivalent chemical shifts identical to free S-methylmethionine, demonstrate that both residues are freely exposed to solvent and in an equivalent environment to one another, whereas in the native molecule, the two methionine residues are in very different environments inside the protein and not freely exposed to solvent, as shown by very different chemical shifts and line broadening in NMR (20,21). Surprisingly, this form of denatured myoglobin stimulated clone 14.5 under conditions of chloroquine treatment that inhibited presentation of native myoglobin (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Apomyoglobin was prepared by titration in water to pH 1.5 with HCl and extraction of the heme with 2-butanone, followed by extensive dialysis. S-Methylmyoglobin was prepared by reaction of the methionines of apomyoglobin with methyl iodide as described (20,21) and was dialyzed before use to ensure that it was free of any fragments, although none should have been generated in the reaction.…”

Section: Methodsmentioning

confidence: 99%

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Antigen conformation determines processing requirements for T-cell activation

1985

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

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We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin-namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4CI, the cationic ionophore monen-

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Antigen conformation determines processing requirements for T-cell activation

1985

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

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“…Therefore, we compared the inhibition by the antiidiotypic antiserum of anti-Mb antibodies binding to plates coated with either native Mb or the denatured form, S-methyl Mb. Smethyl Mb has been partially denatured and unfolded by the addition of extra methyl groups to the sulfur atoms of hydrophobic methionine residues at positions 55 and 131, introducing positive charges into the hydrophobic core of the molecule, and thus requiring the molecule to unfold to admit solvent (24,25). The denaturation and free exposure of these regions to aqueous solvent, in contrast to native Mb, was demonstrated by ~sC-NMR, which demonstrated identical sharp resonances for the two S-methyl methionine residues equivalent to free S-methyl methionine, in contrast to the distinct chemical shifts observed for the native molecule (24,25).…”

Section: H-2 Influence Onmentioning

confidence: 99%

A major anti-myoglobin idiotype. Influence of H-2-linked Ir genes on idiotype expression.

Kawamura¹,

Kohno²,

Busch³

et al. 1984

The Journal of Experimental Medicine

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A rabbit antiidiotypic antiserum raised against an A.SW IgG1K monoclonal anti-sperm whale myoglobin (Mb) antibody, HAL19, and extensively absorbed with normal mouse immunoglobulin and MOPC 21 (IgG1K), was found to detect a common or major anti-Mb idiotype expressed by some but not all anti-Mb monoclonal antibodies, regardless of immunoglobulin G (IgG) subclass, and by 40-50% of the anti-Mb antibodies in immune serum from five high responder strains of mice representing five different Igh allotypes. It did not inhibit antibodies to three unrelated protein antigens. The fraction of antibodies expressing this idiotype, denoted IdHAL19, was regulated by H-2-linked genes that correlated exactly in four independent haplotypes and an F1 with the known Mb immune response (Ir) genes and may be identical to these. Whereas less than 50% of antibodies from high responder mice were inhibitable by anti-IdHAL19, greater than 80% of antibodies from low responder mice, tested at comparable final antibody concentration, were inhibitable. This result was true for both low responder haplotypes, H-2b (B10) and H-2k (B10.BR). The idiotype was found to be present on antibodies that bound to native Mb but not fragments 1-55 or 132-153 of Mb or a denatured form, S-methyl Mb. This specificity for native Mb paralleled that of the monoclonal idiotype HAL19 itself. Therefore, the production of antibodies specific for native in contrast to denatured Mb was studied in H-2-congenic high and low responder strains. Strikingly, low responders produced antibodies that reacted almost exclusively with the native conformation, whereas a larger proportion of antibodies from high responder mice also reacted with the denatured form, S-methyl Mb. Bypassing of the Ir gene defect by immunization with Mb attached to a carrier, F gamma G, resulted in low responder antisera resembling higher responder sera in both idiotype expression and conformational specificity. The simplest explanation of these results is that H-2-linked Ir genes control antibody fine specificity, which is reflected in the idiotypes of the variable regions expressed. We suggest that low responder mice produce a more limited repertoire of antibodies consisting primarily of IdHAL19-positive antibodies specific for the native conformation of Mb. High responder mice produce a greater diversity of antibodies to Mb, so that the IdHAL19-positive, conformation-specific population represents a smaller proportion of the total. Similarly, the use of carrier-specific helper T cells in low responder mice results in a greater diversity of antibodies, which dilutes out the IdHAL19 subset.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…We felt that the diastereotopic nature of the two fluorine atoms in l -difluoromethionine ( l - S -(difluoromethyl)homocysteine; DFM, 1 ) could be exploited in the investigation of protein structure and function. Although methyl groups in proteins have been shown to have little rotational restriction, the presence of the two fluorine atoms introduces a slightly larger size and shape to the methyl group . This altered size could be useful in exploring the three-dimensional space surrounding the methionyl methyl group, which is difficult to achieve using any of the 19 other standard amino acids or currently available unnatural analogues.…”

Section: Introductionmentioning

confidence: 99%

Difluoromethionine as a Novel ¹⁹F NMR Structural Probe for Internal Amino Acid Packing in Proteins

et al. 1999

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The successful incorporation of difluoromethionine (DFM), a novel 19F NMR probe of internal amino acid packing, into the three methionine positions (1, 14, and 107) of a recombinant protein, the lysozyme from bacteriophage λ (LaL), is reported. The anisochronous 19F NMR signals of the diastereotopic fluorines showed a variation in the degree of chemical shift difference when present at relatively free surface positions (Met1 and Met107) versus the tightly packed protein core (Met14), with the anisochronicity greatly enhanced for DFM incorporated at this latter position. The increased magnetic nonequivalence of the two fluorines at position 14 is thought to be a consequence of the restricted environment of DFM at this position. The anisochronicity of these two fluorines is further manifested in a differential chemical shift change for these two fluorines upon binding of an oligosaccharide inhibitor to LaL, with one of the two fluorines experiencing a significant upfield shift compared to the other. This differential variation is thought to be associated with a very subtle change in the protein conformation surrounding one fluorine at position 14, which is not significantly translated to the environment of the other fluorine.

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Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

Cited by 96 publications

References 66 publications

Antigen conformation determines processing requirements for T-cell activation

Antigen conformation determines processing requirements for T-cell activation

A major anti-myoglobin idiotype. Influence of H-2-linked Ir genes on idiotype expression.

Difluoromethionine as a Novel ¹⁹F NMR Structural Probe for Internal Amino Acid Packing in Proteins

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Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

Cited by 96 publications

References 66 publications

Antigen conformation determines processing requirements for T-cell activation

Antigen conformation determines processing requirements for T-cell activation

A major anti-myoglobin idiotype. Influence of H-2-linked Ir genes on idiotype expression.

Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins

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Difluoromethionine as a Novel ¹⁹F NMR Structural Probe for Internal Amino Acid Packing in Proteins