No abstract

Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel’s gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an “asymmetric” gating behavior, with closure only at one electric field´s orientation depending on the channel’s orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel’s gating behavior.

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded β-barrel.

β-turns are secondary structure elements not only exposed on protein surfaces, but also frequently found to be buried in protein–protein interfaces. Protein engineering so far considered mainly the backbone-constraining properties of synthetic β-turn mimics as parts of surface-exposed loops. A β-turn mimic, Hot═Tap, that is available in gram amounts, provides two hydroxyl groups that enhance its turn-inducing properties besides being able to form side-chain-like interactions. NMR studies on cyclic hexapeptides harboring the Hot═Tap dipeptide proved its strong β-turn-inducing capability. Crystallographic analyses of the trimeric fibritin-foldon/Hot═Tap hybrid reveal at atomic resolution how Hot═Tap replaces a βI’-turn by a βII’-type structure. Furthermore, Hot═Tap adapts to the complex protein environment by participating in several direct and water-bridged interactions across the foldon trimer interface. As building blocks, β-turn mimics capable of both backbone and side-chain mimicry may simplify the design of synthetic proteins.