Probing protein - DNA interaction by single molecule and structural analysis

In proteins, conformational change impacting their function has been well investigated in the past decades, and was named `allosteric effect'. However, in DNA-protein interaction, the concept of DNA conformational change caused by DNA-protein binding will affect another nearby DNA-binding protein has not been well investigated and understood. Combined with structural biology and Single Molecule Assays, we can now probe and study allosteric propagation through DNA which exists as a fundamental property in DNA-protein interaction, and this allosteric effect through DNA can fine tune gene expression. Therefore, DNA conformational changes should be seriously considered and analyzed for DNA –protein interactions in general.

Download Full-text

Mapping protein binding stability on nucleosomal DNA by single-molecule approach

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409888x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C111-C111

Author(s):

Jianshi Jin ◽

Teng-fei Lian ◽

Xiaoliang Xie ◽

Xiao-Dong Su

Keyword(s):

Dna Binding ◽

Single Molecule ◽

Conformational Changes ◽

Protein Interaction ◽

Binding Sites ◽

Binding Domain ◽

Shielding Effect ◽

Dna Binding Domain ◽

Nucleosomal Dna ◽

Dna Protein Interaction

The conformation of nucleosomal DNA is significantly different from that of a canonical B-form double stranded DNA (dsDNA), and is generally regarded to be less flexible and less accessible than free dsDNA due to the tight association of histone cores. Previous studies have demonstrated that the key mechanism involved in nucleosomal DNA-protein interaction is the protein accessibility to the DNA binding site. In this work, we used single molecule assays to measure the stability of two transcriptional factors (glucocorticoid receptor DNA binding domain (GRDBD) and estrogen receptor DNA-binding domain (ERDBD)) bound to their binding sites on different positions of the nucleosomal DNA. Interestingly, the results demonstrated that the nucleosomal DNA-GRDBD binding is not always consistent with the histone shielding effect, but adjusted by additional structural changes. Furthermore, the changes of these DNA-GRDBD interaction profiles were confirmed using molecular modeling and docking approaches based on their crystal structures. Very differently, ERDBD essentially is unable to bind to the nucleosomal DNA anywhere including the unblocked positions. We thus have concluded that the nucleosomal DNA-protein interaction is regulated not only by the histone shielding of the DNA binding sites, but also by the conformational changes of the nucleosomal DNA.

Download Full-text

Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA–protein interaction

Journal of Magnetism and Magnetic Materials ◽

10.1016/j.jmmm.2008.11.018 ◽

2009 ◽

Vol 321 (7) ◽

pp. 655-658

Author(s):

Hirofumi Kurita ◽

Hachiro Yasuda ◽

Kazunori Takashima ◽

Shinji Katsura ◽

Akira Mizuno

Keyword(s):

Single Molecule ◽

Protein Interaction ◽

Dna Protein Interaction ◽

Physical Manipulation

Download Full-text

Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA–Protein Interaction

Biochemistry ◽

10.1021/acs.biochem.5b01085 ◽

2015 ◽

Vol 54 (51) ◽

pp. 7457-7469 ◽

Cited By ~ 32

Author(s):

Poulami Talukder ◽

Shengxi Chen ◽

Basab Roy ◽

Petro Yakovchuk ◽

Michelle M. Spiering ◽

...

Keyword(s):

Conformational Changes ◽

Protein Interaction ◽

Fluorescent Probes ◽

Protein Conformational Changes ◽

Dna Protein Interaction

Download Full-text

1P327 Development of a novel mechano-fluorescence microscope for measurement of DNA/protein interaction at single molecule level II

Seibutsu Butsuri ◽

10.2142/biophys.45.s113_3 ◽

2005 ◽

Vol 45 (supplement) ◽

pp. S113

Author(s):

H. Yokota ◽

YW. Han ◽

Jean-Francois Allemand ◽

Xueuang Xi ◽

Vincent Croquette ◽

...

Keyword(s):

Single Molecule ◽

Protein Interaction ◽

Fluorescence Microscope ◽

Single Molecule Level ◽

Level Ii ◽

Dna Protein Interaction

Download Full-text

Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses

10.1101/2021.09.07.459295 ◽

2021 ◽

Author(s):

Claire Gething ◽

Joshua Ferrar ◽

Bishal Misra ◽

Giovanni Howells ◽

Ucheor B. Choi

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Synaptic Vesicle ◽

Conformational Change ◽

Single Molecule ◽

Conformational Changes ◽

Snare Complex ◽

Complex Assembly ◽

Ribbon Synapses ◽

Snare Complex Formation

AbstractNeurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

Download Full-text

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽

10.1098/rsob.200406 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Evelyn Ploetz ◽

Gea K. Schuurman-Wolters ◽

Niels Zijlstra ◽

Amarins W. Jager ◽

Douglas A. Griffith ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Conformational Changes ◽

Protein Interactions ◽

Transmembrane Domain ◽

Substrate Binding ◽

Titration Calorimetry ◽

Uptake System ◽

Conformational States ◽

Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

Download Full-text

Nanoscale Analysis of the Effect of Pathogenic Mutations on Polycystin-1

ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2010-13093 ◽

2010 ◽

Author(s):

Liang Ma ◽

Meixiang Xu ◽

Andres F. Oberhauser

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Protein Interactions ◽

Structure And Function ◽

Eukaryotic Cells ◽

Mechanical Forces ◽

Force Microscopy ◽

Physiological Conditions ◽

Atomic Force ◽

And Function

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells is the result of cycles of mechano-sensing, mechano-transduction, and mechano-response. Recently developed single-molecule atomic force microscopy (AFM) techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins, such as titin (an elastic mechano-sensing protein found in muscle) and polycystin-1 (PC1, a mechanosensor found in the kidney).

Download Full-text

Cofactor-mediated amyloidogenesis

Bioscience Reports ◽

10.1042/bsr20190327 ◽

2019 ◽

Vol 39 (3) ◽

Author(s):

Jillian Madine

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Protein Interactions ◽

Zinc Binding ◽

Amyloid Protein ◽

Wide Range ◽

Pathogenesis Related Protein ◽

Plant Pathogenesis

Abstract A recent study published in Bioscience Reports by Sheng et al. (Bioscience Reports, (2019) 39, pii:BSR20182345] described a small but significant conformational change that occurs upon zinc binding and results in initiation of the amyloidogenic aggregation cascade of Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) in the presence of heparin. The present study describes a two-stage process that is required for the initiation of the amyloidogenic aggregation cascade involving a concentration step and a conformation change to enhance accessibility of natively protected amyloidogenic regions for self-association. For GAPR-1 in the present study, these steps are provided by zinc binding causing the required conformational change enhancing accessibility of amyloidogenic regions, and heparin providing a template or scaffold in turn increasing the local protein concentration. Cofactors such as glycosaminoglycans and metal ions have been found associated with amyloid deposits in vivo and shown to affect protein assembly kinetics in vitro. Cofactor interactions with the amyloidogenic process are an area of great interest for therapeutic intervention for the wide range of diseases known to be associated with amyloid protein aggregation. The present study emphasises the need for enhanced structural understanding of cofactor–amyloid protein interactions and highlights that small subtle conformational changes can have large impacts on resulting aggregation processes.

Download Full-text