An AFM-based single-molecule approach shows how the chaperone and insertase YidC stabilizes E. coli LacY in the unfolded state and assists LacY to insert and fold transmembrane structural segments in random order until folding of the native state is complete. How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone and insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and the membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, indicating that LacY can fold along variable pathways toward the native structure. Since YidC is essential in membrane protein biogenesis and LacY is a model for the major facilitator superfamily, our observations have general relevance.

We observe how translocon and insertase insert single polypeptides along remarkably different pathways to fold membrane proteins.

Significance: Lactose permease of Escherichia coli (LacY), a model for the major facilitator superfamily, catalyzes galactopyranoside/H ⁺ symport across the membrane by a mechanism in which large conformational changes expose the sugar-binding site in the middle of the molecule alternatively to either side of the membrane (an alternating access mechanism). Despite substantial progress with respect to static X-ray crystal structures of LacY, the dynamics of the transport mechanism are not fully understood. Here we use dynamic single-molecule force spectroscopy to quantify the structural properties that change upon substrate binding. The results reveal very significant changes in conformational, kinetic, energetic, and mechanical properties primarily in the N-terminal 6-helix bundle, while the C-terminal 6-helix bundle remains largely unaffected.

Covalent grafting of amino groups onto the carboxylic acid functionalities, naturally covering the surface of fluorescent nanoparticles produced from silicon carbide (SiC NPs), allowed tuning of their surface charge from negative to highly positive. Incubating 3T3-L1 fibroblast cells with differently charged SiC NPs demonstrates the crucial role of the charge in cell fluorescent targeting. Negatively charged SiC NPs concentrate inside the cell nuclei. Close to neutrally charged SiC NPs are present in both cytoplasm and nuclei while positively charged SiC NPs are present only in the cytoplasm and are not able to move inside the nuclei. This effect opens the door for the use of SiC NPs for easy and fast visualization of long-lasting biological processes taking place in the cell cytosol or nucleus as well as providing a new long-term cell imaging tool. Moreover, here we have shown that the interaction between charged NPs and nuclear pore complex plays an essential role in their penetration into the nuclei.