Biology subjects

Fessl, T.

Publications and source records attributed to Fessl, T..

3 recordsLinked to original sources

bioRxiv2026

Heterotrimeric G proteins exhibit subtype-specific mobility differences in live cells

Heterotrimeric G proteins are key signal transducers in all eukaryotic cells. Anchored to the inner leaflet of the plasma membrane, they transmit and amplify extracellular chemical and physical signals downstream of G protein-coupled receptors. Despite numerous available studies, many biophysical aspects that regulate G protein signaling, including membrane mobility, remain poorly understood. Here, using single-molecule imaging, we show that different subtypes of heterotrimeric G proteins containing G{beta}1{gamma}2 subunits exhibit high diversity in their membrane mobility. We demonstrate that the nature of the G subunit plays a major role in defining the mobility of such heterotrimers. Our results indicate that heterotrimers containing G12 and G13 subunits exhibit markedly reduced mobility compared to those containing Gi/o and Gs subunits. These findings identify subtype-specific lateral membrane mobility of G proteins as a factor that can affect their signaling dynamics in living cells.

biophysics↗

bioRxiv2019

Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation

The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster ([~]20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric coupling in which SecA steers the energy landscape of the protein-channel.

biophysics↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗