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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (31 October 2022) | Viewed by 13905

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Prof. Dr. Yuri Lyubchenko

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Department of Pharmaceutical Sciences, College of Pharmacy, WSH, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA
Interests: nanomedicine; biophysics; DNA replication and recombination; protein-DNA interaction; chromatin structure and dynamics; Alzheimer's disease; Parkinson's disease; amyloid protein aggregation; protein misfolding; alpha-synuclein; beta amyloid; protein–protein interaction; computational modeling; nanoimaging; single-molecule biophysics; force spectroscopy; atomic force microscopy; AFM
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Dr. Mohtadin Hashemi

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Department of Pharmaceutical Sciences, WSH 1025, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA
Interests: molecular biophysics; nanotechnology; AFM; molecular simulations

Special Issue Information

Dear Colleagues,

The 6th Midwest Single Molecule Workshop (https://www.unmc.edu/msmw/index.html) continues the tradition of previous workshops by gathering the leading experts and researchers in single-molecule biophysics from across the Midwest and surrounding areas to share their latest research, discoveries and ideas as well as communicate with peers involved in single molecule studies.

This two-day meeting will feature a wide variety of topics and presenters, including invited lectures, poster presentations, short talks and a keynote address. We hope to cover all aspects of single molecule biophysics, including experimental and theoretical approaches and the application of these methods to single molecule studies ranging from isolated individual molecules to whole cells.

The workshop venue moves between different Midwestern universities. This time, the meeting will take place at UNMC, co-organized by Professor Yuri Lyubchenko and Dr. Mohtadin Hashemi. The past five workshops, held at Washington University in St. Louis (2010), University of Michigan at Ann Arbor (2012), University of Illinois at Urbana Champaign (2014), University of Iowa (2016), and Iowa State University (2018) were very popular and each attracted over 100 participants.

In addition, for this 6th Workshop attendees are encouraged to submit their findings to this Special Issue in the International Journal of Molecular Sciences (https://www.mdpi.com/journal/ijms). Every attendee will receive a substantial discount for submissions to this Special Issue.

We look forward to receiving your submission.

Prof. Dr. Yuri Lyubchenko
Dr. Mohtadin Hashemi
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (7 papers)

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Research

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16 pages, 3163 KiB

Open AccessArticle

Single-Molecule Analysis of the Improved Variants of the G-Quadruplex Recognition Protein G4P

by Paras Gaur, Fletcher E. Bain, Masayoshi Honda, Sophie L. Granger and Maria Spies

Int. J. Mol. Sci. 2023, 24(12), 10274; https://doi.org/10.3390/ijms241210274 - 17 Jun 2023

Viewed by 1800

Abstract

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine–guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein’s affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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19 pages, 5477 KiB

Open AccessArticle

Factors That Control the Force Needed to Unfold a Membrane Protein in Silico Depend on the Mode of Denaturation

by Nabil F. Faruk, Xiangda Peng and Tobin R. Sosnick

Int. J. Mol. Sci. 2023, 24(3), 2654; https://doi.org/10.3390/ijms24032654 - 31 Jan 2023

Viewed by 1199

Abstract

Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane–protein interactions had only moderate influences, except for the extraction of the last helix where the membrane–protein interactions had a stronger influence. The up–down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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11 pages, 2067 KiB

Open AccessArticle

Nanorings to Probe Mechanical Stress of Single-Stranded DNA Mediated by the DNA Duplex

by Karen Zagorski, Tommy Stormberg, Mohtadin Hashemi, Anatoly B. Kolomeisky and Yuri L. Lyubchenko

Int. J. Mol. Sci. 2022, 23(21), 12916; https://doi.org/10.3390/ijms232112916 - 26 Oct 2022

Cited by 1 | Viewed by 1264

Abstract

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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12 pages, 6957 KiB

Open AccessArticle

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

by Zhiqiang Sun, Tommy Stormberg, Shaun Filliaux and Yuri L. Lyubchenko

Int. J. Mol. Sci. 2022, 23(19), 11404; https://doi.org/10.3390/ijms231911404 - 27 Sep 2022

Cited by 3 | Viewed by 1432

Abstract

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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Review

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32 pages, 3681 KiB

Open AccessReview

Unravelling How Single-Stranded DNA Binding Protein Coordinates DNA Metabolism Using Single-Molecule Approaches

by Longfu Xu, Matthew T. J. Halma and Gijs J. L. Wuite

Int. J. Mol. Sci. 2023, 24(3), 2806; https://doi.org/10.3390/ijms24032806 - 01 Feb 2023

Cited by 6 | Viewed by 2597

Abstract

Single-stranded DNA-binding proteins (SSBs) play vital roles in DNA metabolism. Proteins of the SSB family exclusively and transiently bind to ssDNA, preventing the DNA double helix from re-annealing and maintaining genome integrity. In the meantime, they interact and coordinate with various proteins vital for DNA replication, recombination, and repair. Although SSB is essential for DNA metabolism, proteins of the SSB family have been long described as accessory players, primarily due to their unclear dynamics and mechanistic interaction with DNA and its partners. Recently-developed single-molecule tools, together with biochemical ensemble techniques and structural methods, have enhanced our understanding of the different coordination roles that SSB plays during DNA metabolism. In this review, we discuss how single-molecule assays, such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations, have advanced our understanding of the binding dynamics of SSBs to ssDNA and their interaction with other proteins partners. We highlight the central coordination role that the SSB protein plays by directly modulating other proteins’ activities, rather than as an accessory player. Many possible modes of SSB interaction with protein partners are discussed, which together provide a bigger picture of the interaction network shaped by SSB. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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25 pages, 6335 KiB

Open AccessReview

Looking at Biomolecular Interactions through the Lens of Correlated Fluorescence Microscopy and Optical Tweezers

by Anahita Haghizadeh, Mariam Iftikhar, Shiba S. Dandpat and Trey Simpson

Int. J. Mol. Sci. 2023, 24(3), 2668; https://doi.org/10.3390/ijms24032668 - 31 Jan 2023

Viewed by 2691

Abstract

Understanding complex biological events at the molecular level paves the path to determine mechanistic processes across the timescale necessary for breakthrough discoveries. While various conventional biophysical methods provide some information for understanding biological systems, they often lack a complete picture of the molecular-level details of such dynamic processes. Studies at the single-molecule level have emerged to provide crucial missing links to understanding complex and dynamic pathways in biological systems, which are often superseded by bulk biophysical and biochemical studies. Latest developments in techniques combining single-molecule manipulation tools such as optical tweezers and visualization tools such as fluorescence or label-free microscopy have enabled the investigation of complex and dynamic biomolecular interactions at the single-molecule level. In this review, we present recent advances using correlated single-molecule manipulation and visualization-based approaches to obtain a more advanced understanding of the pathways for fundamental biological processes, and how this combination technique is facilitating research in the dynamic single-molecule (DSM), cell biology, and nanomaterials fields. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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18 pages, 4552 KiB

Open AccessReview

Atomic Force Microscopy Reveals Complexity Underlying General Secretory System Activity

by Dylan R. Weaver and Gavin M. King

Int. J. Mol. Sci. 2023, 24(1), 55; https://doi.org/10.3390/ijms24010055 - 20 Dec 2022

Cited by 1 | Viewed by 1720

Abstract

The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bilayers. The association of SecA with SecYEG, forming membrane-bound SecYEG/SecA translocases, is directly visualized. Recent work showing topographical aspects of the translocation process that vary with precursor species is also discussed. The data suggests that the Sec system does not employ a single translocation mechanism. We posit that differences in the spatial frequency distribution of hydrophobic content within precursor sequences may be a determining factor in mechanism selection. Precise AFM investigations of active translocases are poised to advance our currently vague understanding of the complicated macromolecular movements underlying protein export across membranes. Full article

(This article belongs to the Special Issue Recent Advances in Single Molecule Studies)

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