Biology subjects

Tuma, R.

Publications and source records attributed to Tuma, R..

3 recordsLinked to original sources

bioRxiv2018

Hydrogen exchange protection factors can be extracted from sparse HDX-MS data

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (MS) is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time dependent change in mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens but determining the latter from the former is generally not possible. Here we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) can be predicted. In most cases, the solution may not be unique, so a number of solutions have to be considered. Such analysis always provides meaningful constraints on protection factors and thus can be used in situations where obtaining more refined data is impractical, e.g., high throughput structure and interaction fingerprinting. It also provides a systematic way to improve data collection strategies in order to obtain unambiguous information at single residue level, e.g. for assessing protein structure predictions at atomistic level.

biophysics↗

bioRxiv2018

Stability of local secondary structure determines selectivity of viral RNA chaperones

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus NSP2 and avian reovirus {sigma}NS. Here, we compared the abilities of NSP2 and {sigma}NS to mediate sequence-specific interactions between rotavirus genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while {sigma}NS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric {sigma}NS oligomerises into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

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↗