Nanopores: a versatile tool to study protein dynamics

Essays in Biochemistry ◽

10.1042/ebc20200020 ◽

2020 ◽

Author(s):

Sonja Schmid ◽

Cees Dekker

Keyword(s):

Protein Dynamics ◽

Single Molecule ◽

Conformational Changes ◽

Protein Function ◽

Label Free ◽

Cellular Functions ◽

Ensemble Averaging ◽

Time Resolved ◽

Wide Range ◽

Versatile Tool

Abstract Proteins are the active workhorses in our body. These biomolecules perform all vital cellular functions from DNA replication and general biosynthesis to metabolic signaling and environmental sensing. While static 3D structures are now readily available, observing the functional cycle of proteins – involving conformational changes and interactions – remains very challenging, e.g., due to ensemble averaging. However, time-resolved information is crucial to gain a mechanistic understanding of protein function. Single-molecule techniques such as FRET and force spectroscopies provide answers but can be limited by the required labelling, a narrow time bandwidth, and more. Here, we describe electrical nanopore detection as a tool for probing protein dynamics. With a time bandwidth ranging from microseconds to hours, nanopore experiments cover an exceptionally wide range of timescales that is very relevant for protein function. First, we discuss the working principle of label-free nanopore experiments, various pore designs, instrumentation, and the characteristics of nanopore signals. In the second part, we review a few nanopore experiments that solved research questions in protein science, and we compare nanopores to other single-molecule techniques. We hope to make electrical nanopore sensing more accessible to the biochemical community, and to inspire new creative solutions to resolve a variety of protein dynamics – one molecule at a time.

Protein dynamics enables phosphorylation of buried residues in Cdk2/Cyclin A-bound p27

10.1101/2020.03.12.989517 ◽

2020 ◽

Author(s):

João Henriques ◽

Kresten Lindorff-Larsen

Keyword(s):

Protein Phosphorylation ◽

Protein Dynamics ◽

Single Molecule ◽

Protein Function ◽

Cell Cycle Progression ◽

Conformational Dynamics ◽

Protein Structures ◽

Cyclin A ◽

General Role ◽

Wide Range

AbstractProteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays key roles in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, since kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues and do they lead to changed conformational dynamics. We have used the ternary complex between p27, Cdk2 and Cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed, and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multi-site phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function.Significance StatementProtein phosphorylation is a common post-translation modification and is carried out by kinases. While many phosphorylation sites are located in disordered regions of proteins or in loops, a surprisingly large number of modification sites are buried inside folded domains. This observation led us to ask the question of how kinases gain access to such buried residues. We used the complex between p27, a regulator of cell cycle progression, and Cyclin-dependent kinase 2/Cyclin A to study this problem. We hypothesized that transient exposure of buried tyrosines in p27 to the solvent would make them accessible to kinases, explaining how buried residues get modified. We provide an atomic-level description of these dynamic processes revealing how protein dynamics plays a role in regulation.

Ligand-Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free-Towards a Conformational Activity Screening for Drug Discovery

Angewandte Chemie International Edition ◽

10.1002/anie.201802603 ◽

2018 ◽

Vol 57 (31) ◽

pp. 9955-9960 ◽

Cited By ~ 4

Author(s):

Jörn Güldenhaupt ◽

Marta Amaral ◽

Carsten Kötting ◽

Jonas Schartner ◽

Djordje Musil ◽

...

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Label Free ◽

Time Resolved ◽

Activity Screening

LARMD: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor

Briefings in Bioinformatics ◽

10.1093/bib/bbz141 ◽

2019 ◽

Vol 21 (6) ◽

pp. 2206-2218 ◽

Cited By ~ 15

Author(s):

Jing-Fang Yang ◽

Fan Wang ◽

Yu-Zong Chen ◽

Ge-Fei Hao ◽

Guang-Fu Yang

Keyword(s):

Estrogen Receptor ◽

Protein Dynamics ◽

Protein Function ◽

Inflammatory Diseases ◽

Great Difficulty ◽

Vital Role ◽

Site Directed Mutagenesis ◽

Bioinformatic Tools ◽

Wide Range ◽

User Friendly

Abstract Protein dynamics is central to all biological processes, including signal transduction, cellular regulation and biological catalysis. Among them, in-depth exploration of ligand-driven protein dynamics contributes to an optimal understanding of protein function, which is particularly relevant to drug discovery. Hence, a wide range of computational tools have been designed to investigate the important dynamic information in proteins. However, performing and analyzing protein dynamics is still challenging due to the complicated operation steps, giving rise to great difficulty, especially for nonexperts. Moreover, there is a lack of web protocol to provide online facility to investigate and visualize ligand-driven protein dynamics. To this end, in this study, we integrated several bioinformatic tools to develop a protocol, named Ligand and Receptor Molecular Dynamics (LARMD, http://chemyang.ccnu.edu.cn/ccb/server/LARMD/ and http://agroda.gzu.edu.cn:9999/ccb/server/LARMD/), for profiling ligand-driven protein dynamics. To be specific, estrogen receptor (ER) was used as a case to reveal ERβ-selective mechanism, which plays a vital role in the treatment of inflammatory diseases and many types of cancers in clinical practice. Two different residues (Ile373/Met421 and Met336/Leu384) in the pocket of ERβ/ERα were the significant determinants for selectivity, especially Met336 of ERβ. The helix H8, helix H11 and H7-H8 loop influenced the migration of selective agonist (WAY-244). These computational results were consistent with the experimental results. Therefore, LARMD provides a user-friendly online protocol to study the dynamic property of protein and to design new ligand or site-directed mutagenesis.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821207116 ◽

2019 ◽

Vol 116 (23) ◽

pp. 11247-11252 ◽

Cited By ~ 10

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.

Chemical Decorations of “MARs” Residents in Orchestrating Eukaryotic Gene Regulation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.602994 ◽

2020 ◽

Vol 8 ◽

Author(s):

Tanaya Roychowdhury ◽

Samit Chattopadhyay

Keyword(s):

Gene Regulation ◽

Conformational Changes ◽

Protein Interactions ◽

Protein Function ◽

Functional Outcomes ◽

Three Dimensional ◽

Limited Information ◽

Post Translational Modification ◽

Cellular Functions ◽

3D Matrix

Genome organization plays a crucial role in gene regulation, orchestrating multiple cellular functions. A meshwork of proteins constituting a three-dimensional (3D) matrix helps in maintaining the genomic architecture. Sequences of DNA that are involved in tethering the chromatin to the matrix are called scaffold/matrix attachment regions (S/MARs), and the proteins that bind to these sequences and mediate tethering are termed S/MAR-binding proteins (S/MARBPs). The regulation of S/MARBPs is important for cellular functions and is altered under different conditions. Limited information is available presently to understand the structure–function relationship conclusively. Although all S/MARBPs bind to DNA, their context- and tissue-specific regulatory roles cannot be justified solely based on the available information on their structures. Conformational changes in a protein lead to changes in protein–protein interactions (PPIs) that essentially would regulate functional outcomes. A well-studied form of protein regulation is post-translational modification (PTM). It involves disulfide bond formation, cleavage of precursor proteins, and addition or removal of low-molecular-weight groups, leading to modifications like phosphorylation, methylation, SUMOylation, acetylation, PARylation, and ubiquitination. These chemical modifications lead to varied functional outcomes by mechanisms like modifying DNA–protein interactions and PPIs, altering protein function, stability, and crosstalk with other PTMs regulating subcellular localizations. S/MARBPs are reported to be regulated by PTMs, thereby contributing to gene regulation. In this review, we discuss the current understanding, scope, disease implications, and future perspectives of the diverse PTMs regulating functions of S/MARBPs.

Real Time de novo Deposition of Centromeric Histone-associated Proteins Using the Auxin Inducible Degradation system

10.1101/304725 ◽

2018 ◽

Author(s):

Sebastian Hoffmann ◽

Daniele Fachinetti

Keyword(s):

Real Time ◽

Protein Dynamics ◽

Protein Function ◽

Genome Engineering ◽

De Novo ◽

Protein Complexes ◽

Versatile Tool ◽

Associated Proteins ◽

Endogenous Proteins ◽

Set Up

i.Summary/AbstractMeasuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the Auxin Inducible Degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and re-expression in the same experimental set-up. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and re-activation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

10.1101/476432 ◽

2018 ◽

Author(s):

Michael C. Thompson ◽

Benjamin A. Barad ◽

Alexander M. Wolff ◽

Hyun Sun Cho ◽

Friedrich Schotte ◽

...

Keyword(s):

Protein Dynamics ◽

Conformational Changes ◽

Thermal Excitation ◽

Relaxation Processes ◽

Spectroscopic Techniques ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

Solvent Molecules ◽

Ray Scattering

AbstractCorrelated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the μs-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

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

10.1101/727503 ◽

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.

Computational Tool for Ensemble Averaging of Single-Molecule Data

10.1101/2020.08.10.241752 ◽

2020 ◽

Author(s):

Thomas Blackwell ◽

W. Tom Stump ◽

Sarah R. Clippinger ◽

Michael J. Greenberg

Keyword(s):

Brownian Motion ◽

User Interface ◽

Single Molecule ◽

Conformational Changes ◽

Graphical User Interface ◽

Molecular Motions ◽

Computational Tool ◽

Ensemble Averaging ◽

Mechanochemical Coupling ◽

Kinetics Of

AbstractMolecular motors couple chemical transitions to conformational changes that perform mechanical work in a wide variety of biological processes. Disruption of this coupling can lead to diseases, and therefore there is a need to accurately measure mechanochemical coupling in motors in both health and disease. Optical tweezers, with nanometer spatial and millisecond temporal resolution, have provided valuable insights into these processes. However, fluctuations due to Brownian motion can make it difficult to precisely resolve these conformational changes. One powerful analysis technique that has improved our ability to accurately measure mechanochemical coupling in motor proteins is ensemble averaging of individual trajectories. Here, we present a user-friendly computational tool, Software for Precise Analysis of Single Molecules (SPASM), for generating ensemble averages of single-molecule data. This tool utilizes several conceptual advances, including optimized procedures for identifying single-molecule interactions and the implementation of a change point algorithm, to more precisely resolve molecular transitions. Using both simulated and experimental data, we demonstrate that these advances allow for accurate determination of the mechanics and kinetics of the myosin working stroke with a smaller set of data. Importantly, we provide our open source MATLAB-based program with a graphical user interface that enables others to readily apply these advances to the analysis of their own data.Statement of SignificanceSingle molecule optical trapping experiments have given unprecedented insights into the mechanisms of molecular machines. Analysis of these experiments is often challenging because Brownian motion-induced fluctuations introduce noise that can obscure molecular motions. A powerful technique for analyzing these noisy traces is ensemble averaging of individual binding interactions, which can uncover information about the mechanics and kinetics of molecular motions that are typically obscured by Brownian motion. Here, we provide an open source, easy-to-use computational tool, SPASM, with a graphical user interface for ensemble averaging of single molecule data. This computational tool utilizes several conceptual advances that significantly improve the accuracy and resolution of ensemble averages, enabling the generation of high-resolution averages from a smaller number of binding interactions.

A Perspective on Molecular Structure and Bond-Breaking in Radiation Damage in Serial Femtosecond Crystallography

Crystals ◽

10.3390/cryst10070585 ◽

2020 ◽

Vol 10 (7) ◽

pp. 585 ◽

Cited By ~ 2

Author(s):

Carl Caleman ◽

Francisco Jares Junior ◽

Oscar Grånäs ◽

Andrew V. Martin

Keyword(s):

Radiation Damage ◽

Protein Dynamics ◽

Conformational Changes ◽

Bond Breaking ◽

Time Resolved ◽

Metal Centers ◽

Disulfide Bonding ◽

X Ray ◽

Sample Structure ◽

Effects Of Radiation

X-ray free-electron lasers (XFELs) have a unique capability for time-resolved studies of protein dynamics and conformational changes on femto- and pico-second time scales. The extreme intensity of X-ray pulses can potentially cause significant modifications to the sample structure during exposure. Successful time-resolved XFEL crystallography depends on the unambiguous interpretation of the protein dynamics of interest from the effects of radiation damage. Proteins containing relatively heavy elements, such as sulfur or metals, have a higher risk for radiation damage. In metaloenzymes, for example, the dynamics of interest usually occur at the metal centers, which are also hotspots for damage due to the higher atomic number of the elements they contain. An ongoing challenge with such local damage is to understand the residual bonding in these locally ionized systems and bond-breaking dynamics. Here, we present a perspective on radiation damage in XFEL experiments with a particular focus on the impacts for time-resolved protein crystallography. We discuss recent experimental and modelling results of bond-breaking and ion motion at disulfide bonding sites in protein crystals.