S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding

Alegre-Cebollada, Jorge; Kosuri, Pallav; Giganti, David; Eckels, Edward C.; Rivas‐Pardo, Jaime Andrés; Hamdani, Nazha; Warren, Chad M.; Solaro, R. John; Linke, Wolfgang A.; Fernández, Julio M.

doi:10.1016/j.cell.2014.01.056

Cited by 181 publications

(252 citation statements)

References 67 publications

(71 reference statements)

Supporting

Mentioning

236

Contrasting

Unclassified

Order By: Relevance

“…To differentiate between the two populations, we implemented refolding experiments using a linear increasing ramp in the force (100 pN·s −1 ), or "force ramp." This technique allows us to discern the relative mechanical stability of domains in the denature and probe pulses by measuring the force at which they unfold on the ramp (27). In a representative recording of a force-ramp refolding experiment, a single FimA polyprotein is subject to three unfolding/refolding cycles alternating 1-s and 20-s quench pulses (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.bacterial adhesion | mechanical stability | single-molecule force spectroscopy | Gram-positive pili | isopeptide bond B acterial infections of solid tissues begin with the attachment of bacteria to target surfaces. In many instances, bacteria adhere against forces that oppose such attachment: micturition in the genitourinary tract (1) or mucociliary flow in the respiratory tract (2), for example. In such environments, a completely immobile adherent bacterium experiences a drag force that can be approximated by Stokes law, F = 6·π·r·η·v, where r is the Stokes radius of the bacterium (∼0.5 μm), η is the viscosity of the fluid (in the respiratory mucus, 1-100 Pa·s −1 ) (3), and v is the velocity of the fluid surrounding the bacterium (Fig. 1A). Under normal mucociliary flow (1-100 μm·s −1 ) (4), forces on a single bacterium can exceed several nanonewtons. Such high forces are sufficient to cleave covalent bonds within the initial adherence structures (5), which would terminate attachment. Understanding how bacteria manage to remain attached under such strong mechanical perturbations is of fundamental interest and could identify new targets for antibiotic development.The initial interaction between bacteria and the host is mediated by micrometer-long adhesive structures termed pili or fimbriae (Fig. 1A). Due to their adhesive role, pili are virulence factors that contribute to the development of infections (6). Structurally, pili are polymers of tens to hundreds of subunits, termed shaft pilins, that are assembled in series and are presented at the extracellular surface, often with inclusion of minor pilins that can have adhesive properties (6). Remar...

show abstract

Section: Resultsmentioning

confidence: 99%

“…To measure SpaA and FimA refolding, we applied denaturequench-probe protocols using AFM-based force spectroscopy in "force-clamp" mode (26,27). On the denature pulse, a high force (≥350 pN) is applied to unfold the polyprotein, with individual events observed as ∼12.5-nm steps in the extension vs. time trace.…”

Section: Resultsmentioning

confidence: 99%

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although extremely simple molecular assays such as DNA or RNA hairpins could fit into a single reaction coordinate description [48], increasing slightly the complexity of the molecule leads to a dramatical rise in the complexity of the actual free energy landscape in the system, requiring more detailed studies. In this sense, molecules such as multiple nucleic-acid hairpins [53], protein-ligand complexes [54] or any mechanically pulled protein [55], appear as potential systems where a one-dimensional description takes the risk of leading to a clear misunderstanding of the actual complexity of their conformational space and the dynamical processes to which they are subject.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

Mechanical unfolding of a simple model protein goes beyond the reach of one-dimensional descriptions

Tapia‐Rojo

Arregui

Mazo

et al. 2014

The Journal of Chemical Physics

View full text Add to dashboard Cite

We study the mechanical unfolding of a simple model protein. The Langevin dynamics results are analyzed using Markov-model methods which allow to describe completely the configurational space of the system. Using transition path theory we also provide a quantitative description of the unfolding pathways followed by the system. Our study shows a complex dynamical scenario. In particular, we see that the usual one-dimensional picture: free-energy vs end-to-end distance representation, gives a misleading description of the process. Unfolding can occur following different pathways and configurations which seem to play a central role in one-dimensional pictures are not the intermediate states of the unfolding dynamics.

show abstract

“…3). Intradomain spectroscopic probes showed that the C1 domain exhibits some degree of folding-related dynamics, as is typical of Ig domains in muscle and similar to those found in titin (29).…”

Section: N-terminal Cmybp-c Becomes More Compact and Less Disorderedmentioning

confidence: 99%

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Colson

Thompson

Espinoza-Fonseca

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electronelectron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. muscle | protein kinase A | fluorescence resonance energy transfer | DEER | molecular dynamics simulation

show abstract

S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding

Cited by 181 publications

References 67 publications

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Mechanical unfolding of a simple model protein goes beyond the reach of one-dimensional descriptions

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Contact Info

Product

Resources

About