Structure-activity relationship of the ring portion in backbone-cyclic C-terminal hexapeptide analogs of substance P. NMR and molecular dynamics

Behrens, Stefan; Mathä, Barbara; Bitan, Gal; Gilon, Chaim; Kessler, Horst

doi:10.1111/j.1399-3011.1996.tb00876.x

Cited by 8 publications

(2 citation statements)

References 23 publications

(7 reference statements)

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“…The analyses were carried out in pure or a large excess of DMSO- d 6 , which has been shown to be a close mimic of the aqueous physiological environment, , since the amide region of the spectrum underwent exchange with water under the experimental conditions and the amide signals were broadened out. All samples were prepared in NMR tubes under inert conditions in an oxygen-free M. Braun glovebox to avoid Cu(I) oxidation.…”

Section: Experimental Sectionmentioning

confidence: 99%

Peptide Models of Cu(I) and Zn(II) Metallochaperones: The Effect of pH on Coordination and Mechanistic Implications

2013

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The first NMR structures of Cu(I) and Zn(II) peptide complexes as models of metallochaperones were derived with no predetermined binding mode. The cyclic peptide MDCSGCSRPG was reacted with Cu(I) and Zn(II) at low and moderate pH. This peptide features the conserved sequence of copper chaperones but with Asp at position 2 as appears in the zinc binding domain of ZntA. The structures were compared with those of the Cu(I) complexes of the wild-type sequence peptide MTCSGCSRPG. All analyses were conducted first with no metal-binding constraints to ensure accurate binding ligand assignment. Several structures included metal-Met binding, raising a possible role of Met in the metal transport mechanism. Both Cu(I) and Zn(II) gave different complexes when reacted with the peptide of the native-like sequence under different pH conditions, raising the possibility of pH-dependent transport mechanisms. Cu(I) bound the MTCSGCSRPG peptide through one Cys and the Met under acidic conditions and differently under basic conditions; Zn(II) bound the MDCSGCSRPG peptide through two Cys and the Met residues under acidic conditions and through one Cys and the Met under basic conditions, while Cu(I) bound the non-native Asp mutant peptide through the Asp and one Cys under both conditions, suggesting that Asp may inhibit pH-dependent binding for Cu(I). NOESY and ESI-HRMS supported the presence of an aqua ligand for Zn(II), which likely deprotonated under basic conditions to give a hydroxo group. Coordination similarities were detected among the model system and native proteins, which overall suggest that coordination flexibility is required for the function of metallochaperones.

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Section: Experimental Sectionmentioning

confidence: 99%

Peptide Models of Cu(I) and Zn(II) Metallochaperones: The Effect of pH on Coordination and Mechanistic Implications

2013

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“…1. Apo-sample: Dissolve approximately 1-2 mg of the peptide in 450-500 µl of deuterated NMR grade solvent (the preferable solutions for biological samples are 10% D2O in water or d6-DMSO [11][12] if the sample does not dissolve in water or reacts with it).…”

Section: Sample Preparationmentioning

confidence: 99%

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D <sup>1</sup>H NMR

Shoshan

Tshuva

Shalev

2013

JoVE

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Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy. NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

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Structure-activity relationship of the ring portion in backbone-cyclic C-terminal hexapeptide analogs of substance P. NMR and molecular dynamics

Cited by 8 publications

References 23 publications

Peptide Models of Cu(I) and Zn(II) Metallochaperones: The Effect of pH on Coordination and Mechanistic Implications

Peptide Models of Cu(I) and Zn(II) Metallochaperones: The Effect of pH on Coordination and Mechanistic Implications

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D <sup>1</sup>H NMR

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