Biology subjects

Bilgen, E.

Publications and source records attributed to Bilgen, E..

3 recordsLinked to original sources

bioRxiv2024

Resolving the Dynamic Interplay of the Conformational States in the TPR Domain of Human AMPylase FicD

The human Fic-enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum (ER): FicD reversibly modulates BiPs activity through attaching an adenosine monophosphate (AMP) to the substrate binding domain. This reduces BIPs chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, ATP-bound, and BiP-bound states revealed significant conformational variability in the tetratricopeptide repeat (TPR) motifs. In this study, we investigate the conformational dynamics of FicDs TPR motifs using two-color single-molecule Forster resonance energy transfer (smFRET). We demonstrate that the TPR motifs exhibit conformational dynamics between the TPR-out and TPR-in conformations on timescales ranging from milliseconds to microseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis (PDA) for the FRET constructs and propose a conformational landscape model for FicD where the TPR-in/out states exist in an equilibrium that is altered due to the presence of ATP and BiP.

biophysics↗

bioRxiv2024

Triple labeling resolves a GPCR intermediate state by 3-color single molecule FRET

The correlation of individual conformational changes in dynamic protein complexes remains challenging as most structural methods rely on averaged information over large numbers molecules. Single molecule FRET is a powerful tool for monitoring such conformational changes. When performed using three distinct probes, it enables the correlation of domain movements by providing up to three simultaneous distance measurements with high temporal resolution. Nevertheless, a major challenge lies in the site-specific attachment of three probes to unique positions within the target protein. Here, we propose an orthogonal triple-labeling strategy that is not compromised by native, reactive amino acid functionalities. It combines genetic code expansion and biorthogonal labeling of two different non-canonical amino-acids with an enzymatic self-labeling SNAP tag. We demonstrate its application by establishment of a 3-color sensor on the human metabotropic glutamate receptor 2, a dimeric, multidomain G protein-coupled neuroreceptor, and describe a previously unknown conformational intermediate state using 3-color single molecule FRET.

biophysics↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗