Measurements of the Self-Assembly Kinetics of Individual Viral Capsids Around Their RNA Genome

The formation of a viral capsid-the highly—ordered protein shell that surrounds the genome of a virus—is the canonical example of self-assembly1. The capsids of many positive-sense RNA viruses spontaneously assemble from in vitro mixtures of the coat protein and RNA2. The high yield of proper capsids that assemble is remarkable, given their structural complexity: 180 identical proteins must arrange into three distinct local configurations to form an icosahedral capsid with a triangulation number of 3 (T = 3)1. Despite a wealth of data from structural studies3–5 and simulations6–10, even the most fundamental questions about how these structures assemble remain unresolved. Experiments have not determined whether the assembly pathway involves aggregation or nucleation, or how the RNA controls the process. Here we use interferometric scattering microscopy11,12 to directly observe the in vitro assembly kinetics of individual, unlabeled capsids of bacteriophage MS2. By measuring how many coat proteins bind to each of many individual MS2 RNA strands on time scales from 1 ms to 900 s, we find that the start of assembly is broadly distributed in time and is followed by a rapid increase in the number of bound proteins. These measurements provide strong evidence for a nucleation-and-growth pathway. We also find that malformed structures assemble when multiple nuclei appear on the same RNA before the first nucleus has finished growing. Our measurements reveal the complex assembly pathways for viral capsids around RNA in quantitative detail, including the nucleation threshold, nucleation time, growth time, and constraints on the critical nucleus size. These results may inform strategies for engineering synthetic capsids13 or for derailing the assembly of pathogenic viruses14.

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Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909223116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22485-22490 ◽

Cited By ~ 12

Author(s):

Rees F. Garmann ◽

Aaron M. Goldfain ◽

Vinothan N. Manoharan

Keyword(s):

Self Assembly ◽

Failure Modes ◽

The Self ◽

Coat Proteins ◽

Viral Capsids ◽

Functional Nanostructures ◽

Assembly Kinetics ◽

Collective Phenomenon ◽

Assembly Pathways ◽

Kinetics Of

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

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Temperature-dependent Self assembly of biofilaments during red blood cell sickling

10.1101/2021.03.07.433773 ◽

2021 ◽

Author(s):

Arabinda Behera ◽

Oshin Sharma ◽

Debjani Paul ◽

Anirban Sain

Keyword(s):

Self Assembly ◽

Kinetic Monte Carlo ◽

Lag Time ◽

Vital Role ◽

Temperature Dependent ◽

Opposite Case ◽

Assembly Kinetics ◽

The Mean ◽

Kinetics Of

Molecular self-assembly plays vital role in various biological functions. However, when aberrant molecules self-assemble to form large aggregates, it can give rise to various diseases. For example, the sickle cell disease and Alzheimer’s disease are caused by self-assembled hemoglobin fibers and amyloid plaques, respectively. Here we study the assembly kinetics of such fibers using kinetic Monte-Carlo simulation. We focus on the initial lag time of these highly stochastic processes, during which self-assembly is very slow. The lag time distributions turn out to be similar for two very different regimes of polymerization, namely, a) when polymerization is slow and depolymerization is fast, and b) the opposite case, when polymerization is fast and depolymerization is slow. Using temperature dependent on- and off-rates for hemoglobin fiber growth, reported in recent in-vitro experiments, we show that the mean lag time can exhibit non-monotonic behaviour with respect to change of temperature.

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Faculty Opinions recommendation of Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736685340.793576294 ◽

2020 ◽

Author(s):

Lois Pollack

Keyword(s):

Self Assembly ◽

The Self ◽

Viral Capsids ◽

Assembly Kinetics ◽

Rna Genome ◽

Kinetics Of

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The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins

Biochemical Journal ◽

10.1042/bj2270439 ◽

1985 ◽

Vol 227 (2) ◽

pp. 439-455 ◽

Cited By ~ 15

Author(s):

P M Bayley ◽

F M M Butler ◽

D C Clark ◽

E J Manser ◽

S R Martin

Keyword(s):

Self Assembly ◽

Protein Concentration ◽

Wavelength Dependence ◽

Electrophoretic Analysis ◽

Slow Phase ◽

Condensation Polymerization ◽

Fast Phase ◽

Microtubule Protein ◽

Kinetics Of

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

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Fast Self-Assembly Kinetics of Quantum Dots and a Dendrimeric Peptide Ligand

Langmuir ◽

10.1021/la301227r ◽

2012 ◽

Vol 28 (21) ◽

pp. 7962-7966 ◽

Cited By ~ 39

Author(s):

Jianhao Wang ◽

Pengju Jiang ◽

Zuoyan Han ◽

Lin Qiu ◽

Cheli Wang ◽

...

Keyword(s):

Quantum Dots ◽

Self Assembly ◽

Peptide Ligand ◽

Assembly Kinetics ◽

Kinetics Of

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The role of sucrose concentration in self-assembly kinetics of high methoxyl pectin

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2018.02.103 ◽

2018 ◽

Vol 112 ◽

pp. 1183-1190 ◽

Cited By ~ 14

Author(s):

Daniela Giacomazza ◽

Donatella Bulone ◽

Pier Luigi San Biagio ◽

Rosamaria Marino ◽

Romano Lapasin

Keyword(s):

Self Assembly ◽

Sucrose Concentration ◽

Assembly Kinetics ◽

Kinetics Of

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Self-Assembly Kinetics of Amphiphilic Dendritic Copolymers

Macromolecules ◽

10.1021/acs.macromol.6b02331 ◽

2017 ◽

Vol 50 (4) ◽

pp. 1657-1665 ◽

Cited By ~ 5

Author(s):

Cuiyun Zhang ◽

You Fan ◽

Yunyi Zhang ◽

Cong Yu ◽

Hongfei Li ◽

...

Keyword(s):

Self Assembly ◽

Assembly Kinetics ◽

Kinetics Of

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Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.7.54 ◽

2016 ◽

Vol 7 ◽

pp. 613-629 ◽

Cited By ~ 29

Author(s):

Claudia Koch ◽

Fabian J Eber ◽

Carlos Azucena ◽

Alexander Förste ◽

Stefan Walheim ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Self Assembly ◽

Building Blocks ◽

Mosaic Disease ◽

Inorganic Materials ◽

High Yield ◽

External Control ◽

Immobilization Of Enzymes

The rod-shaped nanoparticles of the widespread plant pathogentobacco mosaic virus(TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

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