The structure, molecular dynamics, and energetics of centrin–melittin complex

Abstract

Centrin is a calcium binding protein belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for calcium binding protein complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin-MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen-MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I’, I’*, and side chain bands for 13C-Crcen, MLT, and the 13C-Crcen-MLT complex. This approach resulted in the determination of MLT’s increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin-MLT complex formation and the dissociation process. Also, discussed is the first structure of a calcium binding protein-MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLT’s N-terminal end are required for complex formation.

Trifluoroacetic acid as excipient destabilizes melittin causing the selective aggregation of melittin within the centrin-melittin-trifluoroacetic acid complex

“…For MLT-TFA complex , the amide I' band comprised TFA (1673.1 cm −1 ), kink (1664.4 cm −1 ), random coil (1656.1 cm −1 ), 3 10 -helix (1643 cm −1 ), β-sheet contributions (1631.3 cm −1 ), and arginine stretching modes (1606.0 and 1582.4 cm −1 ) for the single arginine residue (R 24 ) within the MLT sequence. The β-sheet at 1631.3 cm −1 has also been reported previously by our group 19 and by Levin's group 33 for MLT in aqueous solution. For 13 C- Cr cen , the amide I'* band rendered the following assignments: CaBS loops (1640.0 cm −1 ), the hinge loops (1636 cm −1 ), π-helix (1624.0 cm −1 ), α-helical contributions (1597.7 cm −1 ), and arginine stretching modes (1611 cm −1 , 1584.7 cm −1 , and 1572 cm −1 ).…”

“…Cr cen is highly stable and has been well characterized in the presence and absence of cations using Fourier transform infrared (FT-IR), 15,16 circular dichroism (CD), 15,17 and Nuclear Magnetic Resonance (NMR) 17,18 spectroscopies. The centrin-MLT complex has been studied by our group and its structure has been determined in the absence of TFA 19 . Protein aggregation at the initial state of the sample prior to performing any crystallization screens may well be the cause of the bottleneck observed in high resolution structure determination of protein-peptide complexes.…”

Relative Stability of Human Centrins and Its Relationship to Calcium Binding

“…32,40−42 A 35 μL aliquot of 60 mg/mL protein in 50 mM HEPES, 150 mM NaCl, 4 mM MgCl 2 , and 4 mM CaCl 2 (pD 6.6) was deposited on a 49 mm × 4 mm custom-milled CaF 2 window with a fixed path length of 40 μm. A reference cell was prepared similarly, and both cells were set in a custom dual-chamber cell holder.…”

“…Therefore, the H → D exchange effectively reduces the number of contributing vibrational modes, without sacrificing structural and dynamic information about the behavior of the protein. 32,40−42,45−50 …”

Dual-Fluorescence l-Amino Acid Reports Insertion and Orientation of Melittin Peptide in Cell Membranes

“…The conformation of the peptide is reported elsewhere. 34 The curved double arrows show the expected orientational freedom for the AFaa fluorophore, where the hydrated species (dotted arrows) are shallower in the bilayer than the nonhydrated ones (solid arrows). We assume that the orientation of transition dipole moment in AFaa fluorophore is the same for these two species.…”