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Article ; Online: The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy.

Cubuk, Jasmine / Stuchell-Brereton, Melissa D / Soranno, Andrea

2022

Abstract: Intrinsically disordered proteins (IDPs) and regions (IDRs) have emerged as key players across many biological functions and diseases. Differently from structured proteins, disordered proteins lack stable structure and are particularly sensitive to ... ...

Abstract	Intrinsically disordered proteins (IDPs) and regions (IDRs) have emerged as key players across many biological functions and diseases. Differently from structured proteins, disordered proteins lack stable structure and are particularly sensitive to changes in the surrounding environment. Investigation of disordered ensembles requires new approaches and concepts for quantifying conformations, dynamics, and interactions. Here, we provide a short description of the fundamental biophysical properties of disordered proteins as understood through the lens of single-molecule fluorescence observations. Single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) provides an extensive and versatile toolbox for quantifying the characteristics of conformational distributions and the dynamics of disordered proteins across many different solution conditions, both in vitro and in living cells.
Language	English
Publishing date	2022-11-23
Publishing country	England
Document type	Journal Article
ISSN	1744-1358 ; 0071-1365
ISSN (online)	1744-1358
ISSN	0071-1365
DOI	10.1042/EBC20220065
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically Disordered Tail.

Stringer, Madison A / Cubuk, Jasmine / Incicco, J Jeremías / Roy, Debjit / Hall, Kathleen B / Stuchell-Brereton, Melissa D / Soranno, Andrea

The journal of physical chemistry. B

2023 Volume 127, Issue 26, Page(s) 5837–5849

Abstract: The cellular milieu is a solution crowded with a significant concentration of different components (proteins, nucleic acids, metabolites, ...

Abstract	The cellular milieu is a solution crowded with a significant concentration of different components (proteins, nucleic acids, metabolites,
MeSH term(s)	Humans ; COVID-19 ; SARS-CoV-2 ; Protein Conformation ; Polyethylene Glycols/chemistry ; RNA ; Nucleocapsid Proteins
Chemical Substances	Polyethylene Glycols (3WJQ0SDW1A) ; RNA (63231-63-0) ; Nucleocapsid Proteins
Language	English
Publishing date	2023-06-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1520-5207
ISSN (online)	1520-5207
DOI	10.1021/acs.jpcb.3c02356
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA.

Cubuk, Jasmine / Alston, Jhullian J / Incicco, J Jeremías / Holehouse, Alex S / Hall, Kathleen B / Stuchell-Brereton, Melissa D / Soranno, Andrea

Nucleic acids research

2023 Volume 52, Issue 5, Page(s) 2609–2624

Abstract: The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA ...

Abstract	The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.
MeSH term(s)	Humans ; Coronavirus Nucleocapsid Proteins/chemistry ; Coronavirus Nucleocapsid Proteins/metabolism ; COVID-19/virology ; Nucleocapsid Proteins/chemistry ; Protein Binding ; RNA, Viral/metabolism ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism
Chemical Substances	Coronavirus Nucleocapsid Proteins ; nucleocapsid phosphoprotein, SARS-CoV-2 ; Nucleocapsid Proteins ; RNA, Viral
Language	English
Publishing date	2023-12-28
Publishing country	England
Document type	Journal Article
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkad1215
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Article ; Online: Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms.

Stuchell-Brereton, Melissa D / Zimmerman, Maxwell I / Miller, Justin J / Mallimadugula, Upasana L / Incicco, J Jeremías / Roy, Debjit / Smith, Louis G / Cubuk, Jasmine / Baban, Berevan / DeKoster, Gregory T / Frieden, Carl / Bowman, Gregory R / Soranno, Andrea

Proceedings of the National Academy of Sciences of the United States of America

2023 Volume 120, Issue 7, Page(s) e2215371120

Abstract: The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of ... ...

Abstract	The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.
MeSH term(s)	Apolipoprotein E4/genetics ; Apolipoprotein E3/chemistry ; Apolipoprotein E2 ; Apolipoproteins E ; Protein Conformation ; Protein Isoforms/metabolism
Chemical Substances	Apolipoprotein E4 ; Apolipoprotein E3 ; Apolipoprotein E2 ; Apolipoproteins E ; Protein Isoforms
Language	English
Publishing date	2023-02-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2215371120
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Article ; Online: The disordered N-terminal tail of SARS CoV-2 Nucleocapsid protein forms a dynamic complex with RNA

Cubuk, Jasmine / Alston, Jhullian J / Incicco, J Jeremías / Holehouse, Alex S / Hall, Kathleen B / Stuchell-Brereton, Melissa D / Soranno, Andrea

bioRxiv

Abstract	The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA Binding Domain (RBD) and its flanking disordered N-Terminal Domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.
Keywords	covid19
Language	English
Publishing date	2023-02-13
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2023.02.10.527914
Database	COVID19

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Article ; Online: Getting control of hydrogel networks with cross-linkable monomers.

Cohn, Pamela G / Qavi, Sahar / Cubuk, Jasmine / Jani, Mihir / Megdad, Mohamed Lamine / Shah, Dhvani / Cattafi, Cara / Baul, Panchatapa / Rajaraman, Shanthi / Foudazi, Reza

Journal of materials chemistry. B

2021 Volume 9, Issue 46, Page(s) 9497–9504

Abstract: The structure of a hydrogel network determines its ability to dissipate stress upon deformation, as well as its ability to swell in water. By designing systems with cross-linkable thiol groups in the monomers, radical thiol-ene chemistry was used to form ...

Abstract	The structure of a hydrogel network determines its ability to dissipate stress upon deformation, as well as its ability to swell in water. By designing systems with cross-linkable thiol groups in the monomers, radical thiol-ene chemistry was used to form controlled networks for acrylamide monomers. The use of radical thiol-ene chemistry effectively suppressed homo-polymerization of the bis(acrylamide) monomer and resulted in networks of alternating thiol and acrylamide monomers. Additionally, if the stoichiometry between the monomers is controlled, the network should approach that of ideality. In the case of bis(acrylamide) monomers, the incorporation of hydrogen-bond donors into the network creates a single network hydrogel with the benefits of high strength and ductility from the simultaneous incorporation of chemical and physical cross-links. Additionally, this strategy suppresses the formation of homo-polymerization in the acrylamide monomer to achieve an alternating network, which is supported with NMR characterization of base-digested fragments. For three different monomer compositions, the resulting gels had high compressive strength (up to 40 MPa) and tunable mechanical properties. The high mechanical strength of the 1 : 1, thiol : ene gel composition is due to the uniform distribution of cross-links, which creates defect-free networks for efficient stress transfer. The present one-pot synthetic strategy toward controlled gel networks affords monomer versatility and synthetic ease, as well as the potential for mechanically robust materials.
MeSH term(s)	Biocompatible Materials ; Hydrogels/chemistry ; Materials Testing ; Mechanical Phenomena ; Molecular Structure ; Rheology
Chemical Substances	Biocompatible Materials ; Hydrogels
Language	English
Publishing date	2021-12-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2702241-9
ISSN	2050-7518 ; 2050-750X
ISSN (online)	2050-7518
ISSN	2050-750X
DOI	10.1039/d1tb00482d
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Article: Getting control of hydrogel networks with cross-linkable monomers

Cohn, Pamela G. / Qavi, Sahar / Cubuk, Jasmine / Jani, Mihir / Megdad, Mohamed Lamine / Shah, Dhvani / Cattafi, Cara / Baul, Panchatapa / Rajaraman, Shanthi / Foudazi, Reza

Journal of materials chemistry B. 2021 Dec. 1, v. 9, no. 46

2021

Abstract	The structure of a hydrogel network determines its ability to dissipate stress upon deformation, as well as its ability to swell in water. By designing systems with cross-linkable thiol groups in the monomers, radical thiol–ene chemistry was used to form controlled networks for acrylamide monomers. The use of radical thiol–ene chemistry effectively suppressed homo-polymerization of the bis(acrylamide) monomer and resulted in networks of alternating thiol and acrylamide monomers. Additionally, if the stoichiometry between the monomers is controlled, the network should approach that of ideality. In the case of bis(acrylamide) monomers, the incorporation of hydrogen-bond donors into the network creates a single network hydrogel with the benefits of high strength and ductility from the simultaneous incorporation of chemical and physical cross-links. Additionally, this strategy suppresses the formation of homo-polymerization in the acrylamide monomer to achieve an alternating network, which is supported with NMR characterization of base-digested fragments. For three different monomer compositions, the resulting gels had high compressive strength (up to 40 MPa) and tunable mechanical properties. The high mechanical strength of the 1 : 1, thiol : ene gel composition is due to the uniform distribution of cross-links, which creates defect-free networks for efficient stress transfer. The present one-pot synthetic strategy toward controlled gel networks affords monomer versatility and synthetic ease, as well as the potential for mechanically robust materials.
Keywords	acrylamides ; compression strength ; crosslinking ; deformation ; hydrogels ; hydrogen bonding ; stoichiometry ; strength (mechanics) ; thiols
Language	English
Dates of publication	2021-1201
Size	p. 9497-9504.
Publishing place	The Royal Society of Chemistry
Document type	Article
ZDB-ID	2702241-9
ISSN	2050-7518 ; 2050-750X
ISSN (online)	2050-7518
ISSN	2050-750X
DOI	10.1039/d1tb00482d
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

Cubuk, Jasmine / Alston, Jhullian J / Incicco, J Jeremías / Singh, Sukrit / Stuchell-Brereton, Melissa D / Ward, Michael D / Zimmerman, Maxwell I / Vithani, Neha / Griffith, Daniel / Wagoner, Jason A / Bowman, Gregory R / Hall, Kathleen B / Soranno, Andrea / Holehouse, Alex S

Nature communications

2021 Volume 12, Issue 1, Page(s) 1936

Abstract: The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom ... ...

Abstract	The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.
MeSH term(s)	Binding Sites ; COVID-19/virology ; Coronavirus Nucleocapsid Proteins/chemistry ; Coronavirus Nucleocapsid Proteins/metabolism ; Dimerization ; Molecular Dynamics Simulation ; Phosphoproteins/chemistry ; Phosphoproteins/metabolism ; Protein Conformation ; Protein Domains ; RNA, Viral/chemistry ; RNA, Viral/metabolism ; SARS-CoV-2/chemistry ; SARS-CoV-2/metabolism
Chemical Substances	Coronavirus Nucleocapsid Proteins ; Phosphoproteins ; RNA, Viral ; nucleocapsid phosphoprotein, SARS-CoV-2
Language	English
Publishing date	2021-03-29
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-21953-3
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

Jasmine Cubuk / Jhullian J. Alston / J. Jeremías Incicco / Sukrit Singh / Melissa D. Stuchell-Brereton / Michael D. Ward / Maxwell I. Zimmerman / Neha Vithani / Daniel Griffith / Jason A. Wagoner / Gregory R. Bowman / Kathleen B. Hall / Andrea Soranno / Alex S. Holehouse

Nature Communications, Vol 12, Iss 1, Pp 1-

2021 Volume 17

Abstract: SARS-CoV-2 nucleocapsid (N) protein is responsible for viral genome packaging. Here the authors employ single-molecule spectroscopy with all-atom simulations to provide the molecular details of N protein and show that it undergoes phase separation with ... ...

Abstract	SARS-CoV-2 nucleocapsid (N) protein is responsible for viral genome packaging. Here the authors employ single-molecule spectroscopy with all-atom simulations to provide the molecular details of N protein and show that it undergoes phase separation with RNA.
Keywords	Science ; Q
Language	English
Publishing date	2021-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

bioRxiv : the preprint server for biology

2020

Abstract: The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom ... ...

Abstract	The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.
Keywords	covid19
Language	English
Publishing date	2020-12-21
Publishing country	United States
Document type	Preprint
DOI	10.1101/2020.06.17.158121
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