scholarly journals Single-molecule observation of ligand binding and conformational changes in FeuA

2019 ◽  
Author(s):  
Marijn de Boer ◽  
Giorgos Gouridis ◽  
Yusran Abdillah Muthahari ◽  
Thorben Cordes
Keyword(s):  
Data Analysis ◽  
Ligand Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Analysis Procedure ◽  
Closed Conformation ◽  
Data Analysis Procedure ◽  
Ligand Interactions ◽  
Substrate Binding Protein

ABSTRACTThe specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10000-fold and temporally stabilize the formed conformation 250-fold.SIGNIFICANCE STATEMENTLigand binding and the coupling of this process to conformational changes in proteins are fundamental to their function. We developed a single-molecule approach that allows the simultaneous observation of ligand binding and conformational changes in the substrate-binding protein FeuA. This allows to directly observe the ligand binding process, ligand-driven conformational changes as well as rare short-lived conformational transitions that are uncoupled from the ligand. These findings provide insight into the fundamental relation between ligand-protein interactions and conformational changes. Our findings are, however, not only of interest to understand protein function, but the developed data analysis procedure allows the determination of (relative) distance changes in single-molecule FRET experiments, for situations in which donor and acceptor fluorophore are influenced by quenching processes.

scholarly journals Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy

2019 ◽  
Vol 116 (23) ◽  
pp. 11247-11252 ◽  
Author(s):  
Toru Kondo ◽  
Jesse B. Gordon ◽  
Alberta Pinnola ◽  
Luca Dall’Osto ◽  
Roberto Bassi ◽  
...  
Keyword(s):  
Correlation Function ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Light Harvesting ◽  
Building Blocks ◽  
Complex Stress ◽  
Correlation Spectroscopy ◽  
Light Harvesting Complex ◽  
Single Molecule Level

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

scholarly journals Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Lucas F. Ribeiro ◽  
Vanesa Amarelle ◽  
Liliane F. C. Ribeiro ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Protein Engineering ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Protein Function ◽  
Cellular Response ◽  
Protein Domain ◽  
Signal Molecule ◽  
Synthetic Protein ◽  
Periplasmic Binding Proteins ◽  
Domain Insertion

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

AN ANALYSIS APPROACH TO ACOUSTIC DETECTION OF EXTENSIVE ATMOSPHERIC SHOWERS

2006 ◽  
Vol 21 (supp01) ◽  
pp. 87-91 ◽  
Author(s):  
DMITRY ZABOROV
Keyword(s):  
Data Analysis ◽  
Acoustic Signal ◽  
Analysis Procedure ◽  
Analysis Approach ◽  
Acoustic Detection ◽  
Baikal Lake ◽  
Air Showers ◽  
Data Analysis Procedure

An experiment related to the search of acoustic signal from air showers has been made at Baikal lake. This report briefly describes the experiment setup, data analysis procedure and obtained results.

scholarly journals A ClyA nanopore tweezer for analysis of functional states of protein-ligand interactions

2019 ◽  
Author(s):  
Xin Li ◽  
Kuohao Lee ◽  
Jianhan Chen ◽  
Min Chen
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Bound States ◽  
Conformational Dynamics ◽  
Electrical Current ◽  
Label Free ◽  
Protein Motions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Molecular Tweezer

AbstractConformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

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

Open Biology ◽  
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.

scholarly journals Photovoice Data Analysis: Critical Approach, Phenomenological Approach, and Beyond

2020 ◽  
Vol 2 (1) ◽  
pp. 136-152 ◽  
Author(s):  
Kwok Kuen TSANG
Keyword(s):  
Social Sciences ◽  
Data Analysis ◽  
Phenomenological Approach ◽  
Analysis Procedure ◽  
Interview Data ◽  
Visual Data ◽  
Critical Approach ◽  
Visual Method ◽  
Methodological Challenges ◽  
Data Analysis Procedure

Photovoice is a visual method that has attracted the attention of researchers in the field of education and social sciences in general. However, there are a number of methodological challenges in photovoice research and one of the challenges facing the researchers is the data analysis procedure. This article proposes a strategy for researchers to handle photovoice data analysis which consists of four stages, including a photograph analysis based on the researcher’s interpretations, a photograph analysis based on the participants’ interpretations, a cross-comparison, and theorization. According to the strategy, researchers should analyze both visual data (participants’ voice) and narrative data (interview data) based on their perspectives as well as those of participants in attempt to generate a more credible visual and narrative explanation and theorization of the phenomenon.

STEREOTIP TERHADAP PEREMPUAN DALAM NOVEL-NOVEL KARYA ABIDAH EL KHALIEQY: TINJAUAN SASTRA FEMINIS

2019 ◽  
Vol 1 (1) ◽  
pp. 29-42
Author(s):  
Yenny Puspita
Keyword(s):  
Data Analysis ◽  
Deep Understanding ◽  
Qualitative Approach ◽  
Analysis Procedure ◽  
Data Sources ◽  
Literary Texts ◽  
Primary Data ◽  
The Novel ◽  
Data Analysis Procedure

This study aims to obtain an overview and deep understanding of the role of women in society, especially the stereotypes of women in the novel Perempuan Berkalung Sorban and Geni Jora by Abidah El Khalieqy. This study uses a descriptive qualitative approach. Primary data sources in this study are novels by Abidah El Khalieqy including: Women Berkalung Sorban (2001) and Geni Jora (2004). Data collection techniques are in the form of document searches. In this case, the form of awareness and flow of feminist thought contained in Indonesian literary texts is understood to mean the use of feminism studies. The data analysis procedure used in this study is content analysis.

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