Characterization of Electrostatic and Nonelectrostatic Components of Protein−Membrane Binding Interactions

Abstract

A general method was developed to determine the thermodynamic parameters for the interaction of protein with membranes. Protein intrinsic tryptophan fluorescence was quenched by titration with large unilamellar vesicles containing 9,10-dibrominated distearoylphosphatidylcholine (Br4-DSPC) or a small amount of trinitrophenylphosphatidylethanolamine (TNP-PE), Binding was modeled as a bimolecular reaction of free protein with a unit of "n" lipid molecules and a dissociation constant, Kd. The contribution of residual fluorescence and light scattering could be eliminated by using the second derivative of the titration function as the basis for calculations. For the binding of C-terminal channel domain polypeptides(178-190 residues) of the colicin El ion channel, n=50-60 and Kd=2-3 nM at pH 4, ionic strength, I=0.12 M, and anionic lipid content = 40% (surface potential, psi o =-30 mV), conditions for which the protein has high activity. Values of n = 95 and 210 for the binding of a C-terminal 293-residue colicin fragment and the 522 residue intact colicin E1 molecule scale qualitatively according to the increase in molecular size. General methods are presented to distinguish the electrostatic (delta G el) and nonelectrostatic (delta G nel) components of the total delta G for binding. Using Br4DSPC as the quencher, the binding of the channel polypeptide, P178, was characterized by delta G approximately -9.8 kcal/mol, and delta G el approximately -7.0 kcal/mol, and delta G el= -2.8 kcal/mol (psi o = -30 mV). Using TNP-PE as the quencher, similar values of delta G approximately -9.3 to -9.9 kcal/mol were determined, a somewhat smaller value for delta G nel approximately -5.0 kcal/mol, and a correspondingly larger value for deltaGnel approximately -4.9 kcal/mol. The existence of a delta G nel component of this magnitude may distinguish proteins that have the potential to insert into the membrane.

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

“…Previously, membrane binding analysis of the p190H 6 revealed that the dissociation constant (K d ) for membrane association of the His-tagged channel peptide did not vary significantly from the untagged, thermolytic channel domain of WT colicin E1. Also, the binding of WT p190H 6 to anionic LUVs under conditions (pH 4, ionic strength, I ϳ 0.1 M, anionic lipid content ϭ 40%) for which the protein has the highest activity was assayed as described earlier (24,31). The observed binding parameters, n ϭ 32-45 (lipid molecules bound per protein) and K d ϭ 0.8 -1.2 nM, are in good agreement with earlier results for the WT colicin thermolytic channel peptide (24,31).…”

Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

“…Finally, changes in entropy (ΔS) and Gibbs free energy (ΔG) can be calculated according to equation [7], which takes into consideration the relationship between the equilibrium constant K and the partition coefficient in terms of the reactant volumes 42 :…”

“…Equation [7] 0.8 being the specific lipid molar volume (L/mol) 42 . Standard deviations are indicated.…”

Membrane Protein Insertion Regulated by Bringing Electrostatic and Hydrophobic Interactions into Play

“…The interplay of hydrophobicity and electrostatics and their distribution within the polypeptide sequence have only been studied in detail for small peptides (3-5, 7, 13-15, 17-20). In the case of proteins, the role of these effects on the association with and the insertion into membranes are still poorly understood (2,(21)(22)(23)(24).The study of the membrane insertion process of the translocation (T) 1 domain of diphtheria toxin (25) can provide precious insight into the interactions between proteins and membranes and the refolding mechanisms of membrane proteins. During intoxication of cells (25), the toxin reaches the early endosomes through the clathrin-coated pathway (26).…”

“…The interplay of hydrophobicity and electrostatics and their distribution within the polypeptide sequence have only been studied in detail for small peptides (3-5, 7, 13-15, 17-20). In the case of proteins, the role of these effects on the association with and the insertion into membranes are still poorly understood (2,(21)(22)(23)(24).…”