Background: How SecYEG opens for co-translational translocation is unknown.Results: Ribosome binding to the SecY complex induces ion channel activity.Conclusion: SecYEG responds to ligand binding by a conformational transition.Significance: Dislocation of the plug prepares entry of the nascent chain.

Background: How SecYEG excludes ions during protein translocation is not known.Results: SecYEG gating is voltage-sensitive.Conclusion: Voltage minimizes the ion leak that is induced by stalling a translocation intermediate.Significance: Preservation of the proton motif force requires voltage-driven conformational changes.

The heterotrimeric SecYEG complex cooperates with YidC to facilitate membrane protein insertion by an unknown mechanism. Here we show that YidC contacts the interior of the SecY channel resulting in a ligand-activated and voltage-dependent complex with distinct ion channel characteristics. The SecYEG pore diameter decreases from 8 Å to only 5 Å for the YidC-SecYEG pore, indicating a reduction in channel cross-section by YidC intercalation. In the presence of a substrate, YidC relocates to the rim of the pore as indicated by increased pore diameter and loss of YidC crosslinks to the channel interior. Changing the surface charge of the pore by incorporating YidC into the channel wall increases the anion selectivity, and the accompanying change in wall hydrophobicity is liable to alter the partition of helices from the pore into the membrane. This could explain how the exit of transmembrane domains from the SecY channel is facilitated by YidC.

The objective of this study was to quantify and compare stretch-mediated responses of primary rat osteoblast-like cells to uniform cyclic strain applied intermittently or continuously. Primary rat osteoblast-like cells were seeded and cultured in silicone rubber dishes for 2 days. They were then subjected to 1000 microstrains at 1 Hz for periods of 60 consecutive minutes or to a series of 15-min stretch followed by 15-min rest, until a total stretch of 60 min. After stretching, cells were incubated and assayed on days 4, 8, 16, and 24 for DNA content, alkaline phosphatase (ALP) activity, and calcium (Ca) content. Additionally, qualitative information was obtained via scanning electron and confocal laser scanning micrographs. Significant increases in DNA were observed for cells stretched intermittently versus cells stretched continuously and versus controls. Results showed significant decreases (p < 0.05) in ALP for cells between stretched groups and between both stretched groups versus controls. Additionally, Ca content was greater in cells stretched intermittently versus controls on days 4 and 8 and versus cells stretched continuously on day 24. In conclusion, intermittently strained cells demonstrated significant decreases in ALP and increases in DNA and Ca versus cells strained continuously. This supports the theory that cells respond to mechanical loading in a "trigger-like" response.

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.