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Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed) Published
Abstract [en]

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE , 2018. Vol. 13, no 6, article id e0198416
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-358704DOI: 10.1371/journal.pone.0198416ISI: 000433900800119PubMedID: 29856839OAI: oai:DiVA.org:uu-358704DiVA, id: diva2:1243367
Funder
EU, European Research Council, 714068Swedish Research Council, VR 2015-04568Knut and Alice Wallenberg Foundation, WAF 2014.0183EU, European Research Council
Note

De tre första författarna delar förstaförfattarskapet.

Available from: 2018-08-31 Created: 2018-08-31 Last updated: 2020-03-19Bibliographically approved
In thesis
1. Orientational dynamics of single molecules applied to protein-DNA target search
Open this publication in new window or tab >>Orientational dynamics of single molecules applied to protein-DNA target search
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells in all life forms regulate their genes with the help of transcription factors (TFs). These proteins turn genes on or off by binding regulatory motifs present in the DNA. Before this binding can occur, a TF needs to search and find the specific binding site in the genome of the cell. TFs have been shown to search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA, where the 1D sliding is essential to speed up the search. The molecular mechanism of the sliding process has been poorly understood, and it has remained a mystery how TFs can manage to both slide fast on non-specific DNA regions but also bind effectively to specific sequences.

To be able to truly understand biophysical processes like this we need to measure the localization and orientation of the interacting single molecules over time. We therefore developed real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS), a method which can resolve molecular kinetics with a 100 times faster time resolution than what can be achieved with conventional single-molecule tracking. Here, we used the method to determine how the transcription factor LacI (lac repressor) explores the DNA surface while searching for its specific target. We simultaneously measured the speed of movement and rotation for fluorescently labeled LacI molecules sliding on flow-stretched DNA. These measurements showed that LacI moves ~40 base pairs (bp) along the DNA for every revolution around it. This pitch is longer than the 10.5 bp period of DNA, which suggests that LacI combines helical groove tracking sliding with frequent slippage, where the protein hops to a nearby major groove. 

To measure the length and frequency of these hops we combined our SMCT-FCS results with measurements of operator bypassing and LacI flipping obtained via single-molecule FRET. This combined analysis showed that LacI hops at most 10-20 bp and does so every 200-700 μs.

By performing stochastic simulations of the sliding process, we show that helical sliding combined with frequent hopping speeds up the overall search process by ~100%, compared to if the protein did not hop.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. p. 75
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1916
Keywords
single molecule imaging, fluorescence correlation spectroscopy, gene regulation, transcription factor, lac operon
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-407065 (URN)978-91-513-0905-7 (ISBN)
Public defence
2020-05-15, Room B8, Biomedicinskt centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2020-04-23 Created: 2020-03-19 Last updated: 2020-05-19

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