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Single Molecule Techniques

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Molecular Diversity".

Deadline for manuscript submissions: closed (15 June 2014) | Viewed by 135188

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Dr. Hans-Heiner Gorris

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Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany
Interests: single molecule analysis in femtoliter arrays; enzyme kinetics; ultrasensitive and background-free detection of analytes; photon-upconverting nanoparticles

Special Issue Information

Dear Colleagues,

Single molecule experiments have unraveled processes that were previously concealed in bulk experiments because they were not synchronized or even heterogeneously distributed among individual molecules in a population. New insights into the function, structure and interactions of individual molecules have been driven by technological advances in single molecule manipulation and detection. Fluorescence spectroscopy is one of the most powerful single molecule techniques because it has a high spatial and temporal resolution and is very sensitive, as long as the background can be efficiently suppressed.

In biochemistry/biophysics, transitions or differences in the conformation of proteins have been observed as dynamic or static heterogeneity. Labeling of proteins and DNA with donor-quencher pairs has enabled the detection of binding interactions by fluorescence resonance energy transfer (FRET). Site-specific labeling of protein subunits has enabled the use of FRET as a nanoscale ruler for measuring conformational transitions. On the other hand, fluorogenic substrates can be employed for investigating the catalytic activity of single enzyme molecules. The analysis of single molecule binding and transition events has entailed a shift from a kinetic to a stochastic perspective on biochemical processes.

This Special Issue of Molecules covers all aspects related to the development and the application of "Single Molecule Techniques". Not only are techniques based on fluorescence spectroscopy welcome but also those based on atomic force spectroscopy, scanning-tunneling electron microscopy or others. It is a pleasure to invite original research as well as review articles that describe and discuss technical developments in the detection and manipulation of single molecules as well as their applications.

Dr. Hans-H. Gorris
Guest Editor

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Keywords

biochemistry/biophysics
conformational dynamics
DNA
dynamic/static heterogeneity
fluorescence resonance energy transfer (FRET)
fluorescence spectroscopy
proteins
single molecules
super-resolution microscopy

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Published Papers (13 papers)

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Editorial

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3 pages, 609 KiB

Open AccessEditorial

Special Issue: Single Molecule Techniques

by Hans H. Gorris

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany

Molecules 2015, 20(5), 7772-7774; https://doi.org/10.3390/molecules20057772 - 28 Apr 2015

Cited by 1 | Viewed by 5425

Abstract

Technological advances in the detection and manipulation of single molecules have enabled new insights into the function, structure and interactions of biomolecules. This Special Issue was launched to account for the rapid progress in the field of “Single Molecule Techniques”. Four original research articles and seven review articles provide an introduction, as well as an in-depth discussion, of technical developments that are indispensable for the characterization of individual biomolecules. Fluorescence microscopy takes center stage in this Special Issue because it is one of the most sensitive and flexible techniques, which has been adapted in many variations to the specific demands of single molecule analysis. Two additional articles are dedicated to single molecule detection based on atomic force microscopy. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

Research

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23 pages, 1981 KiB

Open AccessArticle

Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm

by Henning Höfig^1,2,†, Matteo Gabba^2,*,†, Simón Poblete^3,†, Daryan Kempe¹ and Jörg Fitter^1,2,*

¹ Physikalisches Institut (IA), RWTH Aachen University, Otto-Blumenthal-Straße, 52074 Aachen, Germany

² Institute of Complex Systems (ICS-5), Molecular Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany

³ Institute of Complex Systems (ICS-2), Theoretical Soft Matter and Biophysics, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany

^† These authors contributed equally to this work.

Molecules 2014, 19(12), 19269-19291; https://doi.org/10.3390/molecules191219269 - 25 Nov 2014

Cited by 30 | Viewed by 8782

Abstract

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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18 pages, 3710 KiB

Open AccessArticle

Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories

by Christopher P. Calderon

Ursa Analytics, Denver, CO 80212, USA

Molecules 2014, 19(11), 18381-18398; https://doi.org/10.3390/molecules191118381 - 12 Nov 2014

Cited by 12 | Viewed by 8698 | Correction

Abstract

Optical microscopes and nanoscale probes (AFM, optical tweezers, etc.) afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces numerous challenges due to the complex, crowded, and time changing environments associated with live cells. Fluctuations and spatially varying systematic forces experienced by molecules change over time; these changes are obscured by “measurement noise” introduced by the experimental probe monitoring the system. In this article, we demonstrate how the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS) of Fox et al. [IEEE Transactions on Signal Processing 59] can be used to detect both subtle and abrupt state changes in time series containing “thermal” and “measurement” noise. The approach accounts for temporal dependencies induced by random and “systematic overdamped” forces. The technique does not require one to subjectively select the number of “hidden states” underlying a trajectory in an a priori fashion. The number of hidden states is simultaneously inferred along with change points and parameters characterizing molecular motion in a data-driven fashion. We use large scale simulations to study and compare the new approach to state-of-the-art Hidden Markov Modeling techniques. Simulations mimicking single particle tracking (SPT) experiments are the focus of this study. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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20 pages, 1621 KiB

Open AccessArticle

Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins

by Andreas Hartmann^1,†, Georg Krainer^1,2,† and Michael Schlierf^1,*

¹ B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany

² Molecular Biophysics, University of Kaiserslautern, Kaiserslautern 67663, Germany

^† These authors contributed equally to this work.

Molecules 2014, 19(9), 13735-13754; https://doi.org/10.3390/molecules190913735 - 3 Sep 2014

Cited by 26 | Viewed by 10630

Abstract

Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from ∆G⁰ = −3.6 kJ/mol to ∆G⁰ = −0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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16 pages, 1420 KiB

Open AccessArticle

pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy

by Melanie Köhler¹, Andreas Karner², Michael Leitner², Vesa P. Hytönen^3,4, Markku Kulomaa³, Peter Hinterdorfer^1,2 and Andreas Ebner^1,*

¹ Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria

² Center for Advanced Bioanalysis, Gruberstrasse 40, 4020 Linz, Austria

³ Institute of Biomedical Technology, University of Tampere, FI-33014 Tampere, Finland

⁴ Fimlab Laboratories Ltd., FI-33101 Tampere, Finland

Molecules 2014, 19(8), 12531-12546; https://doi.org/10.3390/molecules190812531 - 18 Aug 2014

Cited by 12 | Viewed by 11794

Abstract

Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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Review

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28 pages, 3574 KiB

Open AccessReview

Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

by Krutika Bavishi¹ and Nikos S. Hatzakis^2,*

¹ Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Center for Synthetic Biology "bioSYNergy", Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark

² Bio-Nanotechnology Laboratory, Department of Chemistry, Nano-Science Center, Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, 2100 Copenhagen, Denmark

Molecules 2014, 19(12), 19407-19434; https://doi.org/10.3390/molecules191219407 - 25 Nov 2014

Cited by 17 | Viewed by 10638

Abstract

The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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42 pages, 2951 KiB

Open AccessReview

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

by Alexander Gust, Adrian Zander, Andreas Gietl, Phil Holzmeister, Sarah Schulz, Birka Lalkens, Philip Tinnefeld and Dina Grohmann^*

Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany

Molecules 2014, 19(10), 15824-15865; https://doi.org/10.3390/molecules191015824 - 30 Sep 2014

Cited by 72 | Viewed by 18109

Abstract

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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29 pages, 3050 KiB

Open AccessReview

Enzyme Molecules in Solitary Confinement

by Raphaela B. Liebherr and Hans H. Gorris^*

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg 93040, Germany

Molecules 2014, 19(9), 14417-14445; https://doi.org/10.3390/molecules190914417 - 12 Sep 2014

Cited by 18 | Viewed by 10809

Abstract

Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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16 pages, 2164 KiB

Open AccessReview

Protein Expression Analyses at the Single Cell Level

by Masae Ohno, Peter Karagiannis and Yuichi Taniguchi^*

Laboratory for Single Cell Gene Dynamics, Quantitative Biology Center, RIKEN Address, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan

Molecules 2014, 19(9), 13932-13947; https://doi.org/10.3390/molecules190913932 - 5 Sep 2014

Cited by 19 | Viewed by 9535

Abstract

The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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21 pages, 14040 KiB

Open AccessReview

Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

by Ilko Bald^1,2,* and Adrian Keller^3,*

¹ Institute of Chemistry—Physical Chemistry, Universität Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam, Germany

² BAM Federal Institute of Materials Research and Testing, Richard-Willstätter Str. 11, D-12489 Berlin, Germany

³ Technical and Macromolecular Chemistry, University of Paderborn, Warburger Str. 100, D-33098 Paderborn, Germany

Molecules 2014, 19(9), 13803-13823; https://doi.org/10.3390/molecules190913803 - 3 Sep 2014

Cited by 41 | Viewed by 13322

Abstract

DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM) which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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34 pages, 6136 KiB

Open AccessReview

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

by Beth L. Haas¹, Jyl S. Matson², Victor J. DiRita³ and Julie S. Biteen^1,*

¹ Department of Chemistry, University of Michigan, Ann Arbor, MI 48019, USA

² Department of Medical Microbiology and Immunology, University of Toledo, Toledo, OH 43606, USA

³ Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48019, USA

Molecules 2014, 19(8), 12116-12149; https://doi.org/10.3390/molecules190812116 - 13 Aug 2014

Cited by 42 | Viewed by 12311

Abstract

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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19 pages, 7038 KiB

Open AccessReview

Tracking Electrons in Biological Macromolecules: From Ensemble to Single Molecule

by Leandro C. Tabares¹, Ankur Gupta², Thijs J. Aartsma² and Gerard W. Canters^2,*

¹ Commissariat à l'Energie Atomique, Institut de Biologie et de Technologies de Saclay, Service de Bioénergétique, Biologie Structurale et Mécanismes (CNRS UMR-8221), Gif-sur-Yvette Cedex 91191, France

² Leiden Institute of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, PO Box 9504, RA Leiden 2300, The Netherlands

Molecules 2014, 19(8), 11660-11678; https://doi.org/10.3390/molecules190811660 - 6 Aug 2014

Cited by 7 | Viewed by 8677

Abstract

Nature utilizes oxido-reductases to cater to the energy demands of most biochemical processes in respiratory species. Oxido-reductases are capable of meeting this challenge by utilizing redox active sites, often containing transition metal ions, which facilitate movement and relocation of electrons/protons to create a potential gradient that is used to energize redox reactions. There has been a consistent struggle by researchers to estimate the electron transfer rate constants in physiologically relevant processes. This review provides a brief background on the measurements of electron transfer rates in biological molecules, in particular Cu-containing enzymes, and highlights the recent advances in monitoring these electron transfer events at the single molecule level or better to say, at the individual event level. Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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Other

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3 pages, 768 KiB

Open AccessCorrection

Correction: Calderon, C.P. Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories. Molecules 19, 18381-18398

by Christopher P. Calderon

Ursa Analytics, Denver, CO 80212, USA

Molecules 2015, 20(2), 2828-2830; https://doi.org/10.3390/molecules20022828 - 9 Feb 2015

Viewed by 4910

Abstract

The author wishes to make the following corrections to paper [1] (doi:10.3390/molecules191118381, website: https://www.mdpi.com/1420-3049/19/11/18381): Full article

(This article belongs to the Special Issue Single Molecule Techniques)

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