NOISE ANALYSIS IN STUDIES OF PROTEIN DYNAMICS AND MOLECULAR TRANSPORT

Understanding the role of noise at cellular and higher hierarchical levels depends on our knowledge of the physical mechanisms of its generation. Conversely, noise is a rich source of information about these mechanisms. Using channel-forming protein molecules reconstituted into artificial 5-nm-thick insulating lipid films, it is possible to investigate noise in single-molecule experiments and to relate its origins to protein function. Recent progress in this field is reviewed with an emphasis on how this experimental technique can be used to study low-frequency protein dynamics, including not only reversible ionization of sites on the channel-forming protein molecule, but also molecular mechanisms of 1/f-noise generation. Several new applications of the single-molecule noise analysis to membrane transport problem are also addressed. Among those is a study on antibiotic translocation across bacterial walls. High-resolution recording of ionic current through the single channel, formed by the general bacterial porin, OmpF, enables us to resolve single-molecule events of antibiotic translocation.

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The Joy of Markov Models—Channel Gating and Transport Cycling Made Easy

The Biophysicist ◽

10.35459/tbp.2019.000125 ◽

2021 ◽

Author(s):

G. Zifarelli ◽

P. Zuccolini ◽

S. Bertelli ◽

M. Pusch

Keyword(s):

Single Molecule ◽

Protein Function ◽

Markov Models ◽

Single Channel ◽

Real Life ◽

Power Spectra ◽

K Channel ◽

Modular Architecture ◽

Gating Currents ◽

Discrete State

ABSTRACT The behavior of ion channels and transporters is often modeled using discrete state continuous-time Markov models. Such models are helpful for the interpretation of experimental data and can guide the design of experiments by testing specific predictions. Here, we describe a computational tool that allows us to create Markov models of chosen complexity and to calculate the predictions on a macroscopic scale, as well on a single-molecule scale. The program calculates steady-state properties (current, state probabilities, and cycle frequencies), deterministic macroscopic and stochastic time courses, gating currents, dwell-time histograms, and power spectra of channels and transporters. In addition, a visual simulation mode allows us to follow the time-dependent stochastic behavior of a single channel or transporter. After a basic introduction into the concept of Markov models, real-life examples are discussed, including a model of a simple K+ channel, a voltage-gated sodium channel, a 3-state ligand-gated channel, and an electrogenic uniporter. In this manner, the article has a modular architecture, progressing from basic to more advanced topics. This illustrates how the MarkovEditor program can serve students to explore Markov models at a basic level but is also suited for research scientists to test and develop models on the mechanisms of protein function.

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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.

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Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation

AJP Cell Physiology ◽

10.1152/ajpcell.00659.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. C75-C85 ◽

Cited By ~ 3

Author(s):

Mingjie Tong ◽

R. Keith Duncan

Keyword(s):

Hair Cells ◽

Molecular Mechanisms ◽

Single Channel ◽

Frequency Tuning ◽

Low Frequency ◽

Bk Channels ◽

Bk Channel ◽

Basilar Papilla ◽

Channel Kinetics ◽

Open Probability

Large-conductance, Ca2+-activated, and voltage-gated potassium channels (BK, BKCa, or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary β1 have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the β-subunit, was used to test for the functional presence of β1. The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of β1 and that the level of activation would exhibit a tonotopic gradient following the expression profile of β1. Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary β-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla.

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smBEVO: A computer vision approach to rapid baseline correction of single-molecule time series

10.1101/2021.11.12.468397 ◽

2021 ◽

Author(s):

Khue Tran ◽

Argha Bandyopadhyay ◽

Marcel P Goldschen-Ohm

Keyword(s):

Computer Vision ◽

Time Series ◽

Single Molecule ◽

Molecular Mechanisms ◽

Markov Models ◽

Active Contours ◽

Low Frequency ◽

Lane Detection ◽

Baseline Correction ◽

Baseline Drift

Single-molecule time series inform on the dynamics of molecular mechanisms that are occluded in ensemble-averaged measures. Amplitude-based methods and hidden Markov models (HMMs) frequently used for interpreting these time series require removal of low frequency drift that can be difficult to completely avoid in real world experiments. Current approaches for drift correction primarily involve either tedious manual assignment of the baseline or unsupervised frameworks such as infinite HMMs coupled with baseline nodes that are computationally expensive and unreliable. Here, we develop an image-based method for baseline correction using techniques from computer vision such as lane detection and active contours. The approach is remarkably accurate and efficient, allowing for rapid analysis of single-molecule time series contaminated with nearly any type of slow baseline drift.

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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.

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Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

10.26434/chemrxiv.9847190 ◽

2019 ◽

Author(s):

Zichen Wang ◽

Huaxun Fan ◽

Xiao Hu ◽

John Khamo ◽

Jiajie Diao ◽

...

Keyword(s):

Single Molecule ◽

Protein Function ◽

Hydrophobic Interactions ◽

Transmembrane Helix ◽

Point Mutations ◽

Salt Bridge ◽

Receptor Activation ◽

Membrane Dynamics ◽

Juxtamembrane Domain ◽

Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

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A role for the fusogen eff-1 in epidermal stem cell number robustness in Caenorhabditis elegans

Scientific Reports ◽

10.1038/s41598-021-88500-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sneha L. Koneru ◽

Fu Xiang Quah ◽

Ritobrata Ghose ◽

Mark Hintze ◽

Nicola Gritti ◽

...

Keyword(s):

Stem Cell ◽

Caenorhabditis Elegans ◽

Cell Fate ◽

Single Molecule ◽

Phenotypic Variability ◽

Low Frequency ◽

Time Lapse ◽

Cell Number ◽

Seam Cell ◽

Neuronal Tissues

AbstractDevelopmental patterning in Caenorhabditis elegans is known to proceed in a highly stereotypical manner, which raises the question of how developmental robustness is achieved despite the inevitable stochastic noise. We focus here on a population of epidermal cells, the seam cells, which show stem cell-like behaviour and divide symmetrically and asymmetrically over post-embryonic development to generate epidermal and neuronal tissues. We have conducted a mutagenesis screen to identify mutants that introduce phenotypic variability in the normally invariant seam cell population. We report here that a null mutation in the fusogen eff-1 increases seam cell number variability. Using time-lapse microscopy and single molecule fluorescence hybridisation, we find that seam cell division and differentiation patterns are mostly unperturbed in eff-1 mutants, indicating that cell fusion is uncoupled from the cell differentiation programme. Nevertheless, seam cell losses due to the inappropriate differentiation of both daughter cells following division, as well as seam cell gains through symmetric divisions towards the seam cell fate were observed at low frequency. We show that these stochastic errors likely arise through accumulation of defects interrupting the continuity of the seam and changing seam cell shape, highlighting the role of tissue homeostasis in suppressing phenotypic variability during development.

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Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

International Journal of Molecular Sciences ◽

10.3390/ijms22052398 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2398

Author(s):

Wooyoung Kang ◽

Seungha Hwang ◽

Jin Young Kang ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Facilitated Diffusion ◽

One Dimensional ◽

Bacterial Transcription ◽

Fluorescence Measurements ◽

Dimensional Diffusion ◽

Single Molecule Studies ◽

Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

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Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aβ42 oligomers

Nature Communications ◽

10.1038/s41467-020-20782-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Francesco Simone Ruggeri ◽

Johnny Habchi ◽

Sean Chia ◽

Robert I. Horne ◽

Michele Vendruscolo ◽

...

Keyword(s):

Small Molecules ◽

Single Molecule ◽

Protein Misfolding ◽

Molecular Mechanisms ◽

Chemical Sensitivity ◽

Incomplete Knowledge ◽

Parkinson’S Diseases ◽

Aggregation Inhibitor ◽

Misfolding Diseases

AbstractSignificant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

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