Soekarjo, MarkEisenhawer, MartinKuhn, AndreasVogel, Horst2006-02-272006-02-272006-02-27199610.1021/bi951087hhttps://infoscience.epfl.ch/handle/20.500.14299/226288WOS:A1996TT37500016For the first time, the std. free energy change, DG Deg, of a membrane-inserting protein with a leader sequence has been detd. exptl., using M13 procoat protein as an example. The partition coeff. for the distribution of the procoat protein between the aq. phase and the membrane phase of preformed lipid vesicles yielded a value of G = 6.5*105 M-1, corresponding to a DG Deg of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coeff. of the M13 coat protein between the aq. and the POPC lipid bilayer phase was detd. to be distinctly lower: G = 1*105 M-1 (DG Deg = -9.3 kcal/mol). Proteinase K digestion expts. have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estd. to be -14.8 kcal/mol. To distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coeff. of the mutant procoat protein OM30R [which contains a pos. charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)] was detd., yielding G = 0.3*105 M-1 (DG Deg = -8.6 kcal/mol). Previously reported in vivo expts. have shown that the OM30R mutant protein is not translocated across Escherichia coli membranes but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to DG Deg, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the cryst. lipid phase. Furthermore, by using lipid bilayers with neg. membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coeffs. of the same order of magnitude as for the phosphatidylcholine lipid membrane. [on SciFinder (R)]text::journal::journal article::research article