Author: JH

Publication: Alternating-laser excitation: single-molecule FRET and beyond

J. Hohlbein, T.D. Craggs, T. Cordes, Chemical Society Reviews, 43, 1156-1171, 2014, [link]

The alternating-laser excitation (ALEX) EShistogramOverviewReducedscheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Förster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables the sorting of fluorescently labelled species based on the number and type of fluorophores present. ALEX also provides a convenient way of accessing the correction factors necessary for determining accurate molecular distances. Here, we provide a comprehensive overview of the concept and current applications of ALEX and we explicitly discuss how to obtain fully corrected distance information across the entire FRET range. We also present new ideas for applications of ALEX which will push the limits of smFRET-based experiments in terms of temporal and spatial resolution for the study of complex biological systems.

Publication: Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion (open access)

J. Hohlbein, L. Aigrain, T.D. Craggs, O. Bermek, O. Potapova, P. Shoolizadeh, N.D.F. Grindley, C.M. Joyce, A.N. Kapanidis, Nature Communications, 4,  2131, 2013, [link], open access

The fidelity of DNA polCaptureymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

News: Welcome to Carel Fijen…

…who joins the group to pursue his PhD thesis. He is going to study DNA polymerases interacting with DNA by employing single-molecule FRET techniques. He graduated in Molecular Life Sciences here at Wageningen University.

News: Welcome to Stefan Hutten and Stef van der Krieken…

…who join the group for their Bachelor thesis. In close cooperation with the Laboratory of Biochemistry, we are going to look into Arabidopsis lines that express transcriptional regulators (Stef van der Krieken, project with Prof. Dr. Dolf Weijers) and plasma membrane steroid receptors (Stefan Hutten, project with Prof. Dr. Sacco de Vries).

News: Hello, good-bye, and single molecules

A big welcome to Shazia Farouq (PhD 2012-01-15_T1B17student in the van Amerongen group)! Shazia will take over some of Andy’s projects, who left the group to pursue new endeavours. Thanks to Andy’s work in the last three months, we are now able to detect single fluorescent molecules on our microscope. Hooray!

Picture: Doubly labelled DNA (dyes: Cy3B and ATTO647N) immobilised on a glass surface. The green detection channel is on the left, red detection channel (FRET channel) on the right.

News: Imaging of fluorescent latex beads

After finishing very late yesterday2012-11-29_Beads_MMImages evening, we proudly present our first image taken with the new setup. Nothing very interesting, just some aggregated, fluorescent latex-beads on a cover slip surface imaged into blue, green and red detection channels of an emCCD camera (after excitation with a green laser). A big thank you goes to John for the laser control software!

Now its time to prepare some buffers, get the oxygen scavenger system working, and image some DNA FRET standards.

News: Arrival in Wageningen

After a short flight from Heathrow to Schiphol and some trains and buses later, Andy and I arrived in Wageningen with all our luggage and cardboard-wrapped bikes. On Monday, we had fun opening the boxes from Thorlabs with parts for our new multi-colour TIRF microscope. Opinions on how soon we will be able to detect single, fluorescent molecules seem to differ quite a bit and a couple of bets have been placed, but we accept the challenge and are hoping to share some images in a few weeks time (and collect our winnings).