Marko Sustarsic

Elucidating the Native Architecture of the YidC: Ribosome Complex

Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxa1/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC:ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex.

Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells.

Single-molecule Förster resonance energy transfer (smFRET) serves as a molecular ruler that is ideally posed to study static and dynamic heterogeneity in living cells. Observing smFRET in cells requires appropriately integrated labeling, internalization and imaging strategies, and significant progress has been made towards that goal. Pioneering studies have demonstrated smFRET detection in both prokaryotic and eukaryotic systems, using both wide-field and confocal microscopies, and have started to answer exciting biological questions. We anticipate that future technical developments will open the door to smFRET for the study of structure, conformational changes and kinetics of biomolecules in living cells.

Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy ( = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates. Significance Statement Most genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

DNA Polymerase Conformational Dynamics and the Role of Fidelity-Conferring Residues: Insights from Computational Simulations

Herein we investigate the molecular bases of DNA polymerase I conformational dynamics that underlie the replication fidelity of the enzyme. Such fidelity is determined by conformational changes that promote the rejection of incorrect nucleotides before the chemical ligation step. We report a comprehensive atomic resolution study of wild type and mutant enzymes in different bound states and starting from different crystal structures, using extensive molecular dynamics (MD) simulations that cover a total timespan of ~5 ms. The resulting trajectories are examined via a combination of novel methods of internal dynamics and energetics analysis, aimed to reveal the principal molecular determinants for the (de)stabilization of a certain conformational state. Our results show that the presence of fidelity-decreasing mutations or the binding of incorrect nucleotides in ternary complexes tend to favor transitions from closed toward open structures, passing through an ensemble of semi-closed intermediates. The latter ensemble includes the experimentally observed ajar conformation which, consistent with previous experimental observations, emerges as a molecular checkpoint for the selection of the correct nucleotide to incorporate. We discuss the implications of our results for the understanding of the relationships between the structure, dynamics, and function of DNA polymerase I at the atomistic level.