The analysis of peptide and protein partitioning in lipid membranes is of high relevance for the understanding of biomembrane function. We used statistical thermodynamics analysis to demonstrate the effect of peptide mixing behavior on heat capacity profiles of lipid membranes with the aim to predict peptide aggregation from c(P)-profiles. This analysis was applied to interpret calorimetric data on the interaction of the antibiotic peptide gramicidin A with lipid membranes. The shape of the heat capacity profiles was found to be consistent with peptide clustering in both gel and fluid phase. Applying atomic force microscopy, we found gramicidin A aggregates and established a close link between thermodynamics data and microscopic imaging. On the basis of these findings we described the effect of proteins on local fluctuations. It is shown that the elastic properties of the membrane are influenced in the peptide environment.

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme's major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones.

The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle-vesicle pairs of different diameter (30-200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30-60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2-10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes.

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