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. 2010 Sep 17;49(39):7030-4.
doi: 10.1002/anie.201002906.

Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin

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Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin

Shadakshara S Puttamadappa et al. Angew Chem Int Ed Engl. .

Erratum in

  • Angew Chem Int Ed Engl. 2011 Jul 25;50(31):6948-9
No abstract available

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Figures

Figure 1
Figure 1
NMR analysis of the backbone dynamic of free and trypsin bound MCoTI-I. (a) NMR {15N,1H}-HSQC spectrum of free MCoTI-I. Chemical shift assignments of the backbone amides are indicated. (b) Overlay of the {15N,1H}-HSQC spectra of free (black) and trypsin bound MCoTI-I (red). Residues with large average amide chemical shift differences between two different states ( > 0.3 ppm) are indicated. Peaks that are broadened in trypsin bound MCoTI-I are indicated by grey circles. (c) Average amide chemical shift difference for all the assigned residues in free and trypsin bound MCoTI-I. Chemical shift difference was calculated as: ΔΩ = [(ΔΩNH2+0.04ΔΩN2)/2]½ , where ΔΩNH and ΔΩN are the changes in the amide proton and nitrogen chemical shifts (ppm), respectively. (d) The order parameter, S2, for the free (black) and the trypsin bound MCoTI-I (red). S2 value is a measure of backbone flexibility and represents the degree of angular restriction of N-H vector in the molecular frame. The MCoTI-I loops are shown on top of panels C and D. Small unassigned peaks in both free and trypsin bound spectra of MCoTI-I are from a minor conformation of the protein due to a known isomerization of the backbone at an Asp-Gly sequence in loop 6 of MCoTI-I.
Figure 2
Figure 2
Trypsin binding to MCoTI-I affects the MCoTI-I backbone dynamics. Ribbon (a) and surface (b) diagrams of the trypsin-MCoTI-I interaction map. Red arabic numbers indicate the positions of the MCoTI-I loops. The MCoTI-I residues with a large chemical shift difference (>0.3 ppm) are in blue. (c) Changes in the MCoTI-I order parameter due to binding to trypsin. Residues with Sf2 - Sb2 > 0.2, where Sf/b2 is the order parameter of the free or trypsin bound MCoTI-I, respectively, are depicted in red. MCoTI-I residues that were broadened in {15N,1H}-HSQC due to binding to trypsin are shown in green. We used a structure of free MCoTI-II (PDB code: 1IB9)[6] to illustrate the changes of MCoTI-I dynamics due to trypsin binding.
Figure 3
Figure 3
ε-NH3+ of Lys4 is protected from fast exchange with the solvent in trypsin bound MCoTI-I. {15N,1H}-HSQC spectra of free (a) and trypsin-bound MCoTI-I (b) were collected at RT with the 15N-carrier position at 82 ppm and 15N rf field strengths of 5.2 kHz for 90° and 180° pulses and 1.2 kHz for composite decoupling during acquisition.
Scheme 1
Scheme 1
Primary structure and disulfide connectivities of MCoTI cyclotides. Blue and yellow connectors represent peptide and disulfide bonds, respectively.

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