Substrate conformational dynamics facilitate 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 DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes.

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.

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.

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.

Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E.

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.