Conformational studies of anthrax polypeptide, subtilis polypeptide, and synthetic poly‐γ‐<scp>L</scp>glutamic acid

Balasubramanian, D.; Kalita, C. C.; Kovács, J.

doi:10.1002/bip.1973.360120513

Cited by 20 publications

(12 citation statements)

References 20 publications

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“…The relationship of the root mean square radius to the hydrodynamic radius in each case also suggests that the molecules are present in a random coil configuration. This finding is consistent with our CD analysis as well as with various literature reports of polyglutamic acids at neutral pH (36,37).…”

Section: Resultssupporting

confidence: 94%

“…The chemical identity and homogeneity of the PGGA preparation were confirmed by NMR and amino acid analysis, and the unambiguous structural determination verified the ␥-carboxyl isopeptide linkage. In agreement with previous reports (13,36), GC-FID analysis showed that Ͼ99% of the polymer was composed of D-glutamic acid. This isomeric homogeneity appears to be a unique characteristic of B. anthracis and has further implications for vaccine antigen development in that others have proposed use of polyglutamic acids derived from bacilli including Bacillus subtilis and Bacillus lichenformis, despite the fact that their capsules are often a mixture of D-and L-isomers (28,36).…”

Section: Discussionsupporting

confidence: 93%

“…In agreement with previous reports (13,36), GC-FID analysis showed that Ͼ99% of the polymer was composed of D-glutamic acid. This isomeric homogeneity appears to be a unique characteristic of B. anthracis and has further implications for vaccine antigen development in that others have proposed use of polyglutamic acids derived from bacilli including Bacillus subtilis and Bacillus lichenformis, despite the fact that their capsules are often a mixture of D-and L-isomers (28,36). The use of HPSEC/MALLS allowed for accurate mass determinations independent of column migration behavior, and the detailed mass studies of crude and purified product were instrumental in both furthering the characterization of this antigen and in demonstrating the effect of size reduction on its immunological profile.…”

Section: Discussionsupporting

confidence: 93%

“…To our knowledge, this is the first reported measurement of hydrodynamic radius for PGGA based on light scattering data. The findings are in agreement with the CD spectra and show the molecular conformation to be that of an extended random coil at neutral pH and low ionic strength, consistent with historic studies employing both CD and optical rotary dispersion analyses (36,37,42). Most interestingly, the hydrodynamic radius did not show any appreciable change at pH 4.5 for the control in the EDC study.…”

Section: Discussionsupporting

confidence: 89%

“…It is well documented that polyglutamic acids can exist in defined structural conformations (36,37,42,43) and that these conformations are sensitive to the effects of pH and ionic strength (28,29,36). It is likely that such extended and regularly repeating structures could be disrupted by random cleavage of polymer chains, particularly when the molecular mass is reduced below a certain critical degree of polymerization.…”

Section: Discussionmentioning

confidence: 99%

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Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by ␥-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free ␣-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-␥-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the ␥-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of ␣-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.

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Section: Resultssupporting

confidence: 94%

Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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Critique of the interpretation of the circular dichroism of unordered polypeptides and proteins

Balasubramanian

1974

Biopolymers

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The circular dichroic (CD) profile characterizing unordered polypeptide chains and denatured proteins is the subject of much current attention. Tiffany and Krimm,' and Rippon and Walton2 have suggested that the doubly dichroic CD spectrum (positive band near 218 nm and a much larger negative band near 200 nm) obtained in ionized polyL-glutamic acid [(Glu),] and in polyl-lysine [(Lys),] chains characterize not an unordered conformation (abbreviated as U), but rather a conformation that contains regions of left-handed 31 helical order called the extended helix (abbreviated as EH). This conformational assignment was based on the similarity of this doubly dichroic CD profile with that of the polyL-proline I1 helix. Potential energy calculations, including ionic terms, on ionized (G~u),,~*( and also on ionized (Gl~-Ala),~ have yielded conformational minima for these ionized polymers at (+, $ ) 6 = 84O, 321", and 74O, 343", respectively, compared to the value 103", 326' for the polypropline I1 helix. These similarities in conformational parameters and CD spectra have led Rippon and Walton2 to conclude that the presence of a positive Cotton effect near 218 nm and a negative one near 200 nm is diagnostic of a polypeptide with 31 helical segmental order, i.e., the E H conformation.On the other hand, several workers'-9 have argued thqt the EH-type CD spectra are in fact representative of unordered structures, and that "much or all the CD data of Tiffany and Krimm may be interpreted fts mixtures of helical and unordered forms, and will not require invocation of a third structural form."8 We had observed earlier9 the ubiquity of the doubly dichroic CD spectra in a variety of polypeptides, both ionized and nonionic, under a variety of conditions. That such EH-type spectra might arise in diverse situations such as charge repulsions, protonation, or solvent binding has been claimed recently by Tiffany and Krirnm.lo However, in two recent important papers, Miller et al." have shown that under the conditions of residual blocking groups, concentration, and ionic strength of the medium, (Glu), is indeed not in the E H form, but is a random coil.Additional credence for the latter point of view is provided by the circular dichroic spectra of poly-&taspartic acid and poly-7-bglutamic acid that we present in Figure 1. These two polymers are variants of the usual poly-a-peptide sequence in that the former has an extra methylene group in the primary structural backbone, while poly-y-bglutamic acid has two methylene groups. Yet, as the figure reveals, both poly-8-lraspartate and poly-y-bglutamate display doubly dichroic spectra similar to those seen in ionized (Glu), or (Lys),. (The CD spectra of the acid forms of these two poly-w-peptides are interpreted in terms of a third Cotton effect, induced on the carboxylic acid chromoph0re.12,'~) Qualitatively similar CD spectra are exhibited by ionized copoly-(ybGluyaminobutyric acid),14 and also by the tertiary butyl esters of both these poly-w-amino acids in hexafluoroisopropanol. The additi...

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Poly(ε‐L‐lysine): Synthesis and conformation

Kushwaha¹,

Mathur²,

Balasubramanian³

1980

Biopolymers

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SynopsisThe synthesis of poly(c-L-lysine) is described. This is a poly(6-amino acid) in which the €-amino group of lysine is condensed with the cu-carboxyl group to produce a chain backbone that is a variant of the usual one seen in proteins and the side chain is the a-amino group. Conformational studies of poly(c-L-lysine) and its t -butyloxycarbonyl derivative suggest the likelihood of a chain order that is formally similar to the antiparallel pleated-sheet conformation of proteins.

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Conformational studies of anthrax polypeptide, subtilis polypeptide, and synthetic poly‐γ‐Lglutamic acid

Cited by 20 publications

References 20 publications

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Critique of the interpretation of the circular dichroism of unordered polypeptides and proteins

Poly(ε‐L‐lysine): Synthesis and conformation

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