Related Experiment Videos
Comparison of 15N- and 13C-determined parameters of mobility in melittin
L Zhu1, F G Prendergast, M D Kemple
1Department of Physics, Indiana University Purdue University, Indianapolis 46202-3273, USA.
Abstract:
Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by 15N and 13C NMR. Specifically, inverse-detected 15N T1 and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 degrees C enriched with 15N at six amide positions and in the Trp19 side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and alpha-helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: tau m, the correlation time for the overall rotation; S2, a site-specific order parameter which is a measure of the amplitude of the internal motion; and tau e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the 15N measurements and from 13C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678-1688]. tau m and tau e values were consistent from data on the two nuclei. In the MLT monomer, S2 values for the backbone N-H and C alpha-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C alpha-H order parameters. The Trp side-chain N-H and C-H order parameters, and tau e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.
Insights
Melittin (MLT) dynamics were studied using NMR. Backbone and Trp side-chain mobilities differ between the disordered monomer and alpha-helical tetramer, revealing insights into MLT
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Melittin (MLT) is a 26-residue cytolytic peptide.
- MLT exists as a disordered monomer in dilute aqueous solution and an alpha-helical tetramer at higher concentrations.
- Understanding MLT's dynamics is crucial for its biological function.
Purpose of the Study:
- To investigate the backbone and tryptophan side-chain mobilities of melittin (MLT).
- To compare the dynamics of the disordered MLT monomer and the alpha-helical MLT tetramer.
- To analyze motional parameters using NMR relaxation data and the Lipari-Szabo model-free formalism.
Main Methods:
- Utilized 15N and 13C Nuclear Magnetic Resonance (NMR) spectroscopy.
- Performed inverse-detected 15N T1 and steady-state Nuclear Overhauser Effect (NOE) measurements.
- Analyzed relaxation data using the Lipari-Szabo model-free formalism to determine motional parameters (tau m, S2, tau e).
Main Results:
- Correlation times (tau m and tau e) were consistent between 15N and 13C NMR data.
- In the MLT monomer, backbone N-H and C alpha-H order parameters (S2) were similar.
- In the MLT tetramer, N-H order parameters were approximately 0.2 units larger than C alpha-H order parameters, while Trp side-chain dynamics were similar across both forms.
Conclusions:
- The study provides insights into the distinct motional characteristics of the MLT monomer and tetramer.
- Differences in backbone mobility between the monomer and tetramer suggest conformational changes upon aggregation.
- Trp19 side-chain dynamics are relatively conserved, irrespective of MLT's oligomeric state.