Separation of <i>cis/trans</i> conformers of human and salmon calcitonin by low temperature capillary electrophoresis

Thunecke, Frank; Fischer, Gunter

doi:10.1002/elps.1150190224

Cited by 14 publications

(6 citation statements)

References 24 publications

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“…In this work, we circumvented this problem by performing differential HDX during separation of proteoforms in solution, followed by conformational characterization of the sequentially eluted 2 H-labeled species in real-time using top-down MS. These species are separated by capillary electrophoresis (CE) according to their differing charge to size ratios, − regardless of their overall deuteration level difference. The small volume of the CE capillary allows the use of fully deuterated reagents as the background solvent, thereby enabling HDX during separation.…”

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confidence: 99%

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

et al. 2019

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Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo-and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2 H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2 H during separation based on their disulfidedependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo-and apo-Mb.

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confidence: 99%

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

et al. 2019

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“…As evident from Figure (A), these different conformations are existing in approximate ratios of (i) ~70:30 for the region Leu4–Leu12 and (ii) ~75:25 for the region Gln20–Arg24. The observation is in close agreement with the previous report where, using low temperature capillary electrophoresis, two/three conformers of sCT (in acidic buffer) have been separated in a ratio of 66:34. The close proximity of Pro23 suggests that the two signals for Thr21, Tyr22, and Arg24 correspond to the trans‐isomer and cis‐isomer of Tyr22–Pro23 bond with the minor population corresponding to cis‐Proline conformational state.…”

Section: Discussionmentioning

confidence: 99%

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Rawat

Kumar

2012

Journal of Peptide Science

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Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H(α) chemical shifts and three bond H(N) -H(α) coupling constants indicated that most of the residues of the peptide are populating the α-helical region of the Ramachandran (ϕ, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10(2) -10(3) s(-1) ), inferred by the multiple chemical shift assignments in the region Leu4-Leu12 and around Pro23 (for residues Gln20-Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self-association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The (15) N NMR-relaxation data revealed (i) the presence of slow (microsecond-to-millisecond) timescale dynamics in the N-terminal region (Cys1-Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond-to-picosecond) motions in the C-terminal arm. Put together, the various results suggested that (i) the flexible C-terminal of sCT (from Thr25-Thr31) is involved in identification of specific target receptors, (ii) whereas the N-terminal of sCT (from Cys1-Gln20) in solution - exhibiting significant amount of conformational plasticity and strong bias towards biologically active α-helical structure - facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs.

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“…Capillary zone electrophoresis (CZE) has the potential of serving as a probe for PPIase activity because separation of conformational isomers is possible at low temperatures, as was already shown for the calcium regulatory hormone calcitonin. (Thunecke and Fischer 1998).…”

Section: Monitoring Peptide Bond Cis/trans Isomerase Activitymentioning

confidence: 95%

Regulation of peptide bond cis/trans isomerization by enzyme catalysis and its implication in physiological processes

Fischer

Aumüller

Reviews of Physiology, Biochemistry and Pharmacology

215

150

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In some cases, the slow rotational movement underlying peptide bond cis/trans isomerizations is found to control the biological activity of proteins. Peptide bond cis/trans isomerases as cyclophilins, Fk506-binding proteins, parvulins, and bacterial hsp70 generally assist in the interconversion of the polypeptide substrate cis/trans isomers, and rate acceleration is the dominating mechanism of action in cells. We present evidence disputing the hypothesis that some of the molecular properties of these proteins play an auxiliary role in enzyme function.

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Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis

Cited by 14 publications

References 24 publications

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Regulation of peptide bond cis/trans isomerization by enzyme catalysis and its implication in physiological processes

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