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Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

ABSTRACT: To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 ?m/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ?-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

SUBMITTER: Oude Vrielink AS

PROVIDER: S-EPMC5380327 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

Oude Vrielink Anneloes S AS Vance Tyler D R TD de Jong Arthur M AM Davies Peter L PL Voets Ilja K IK

PloS one 20170404 4

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 μm/s, the MpAFP extender domain fail ...[more]

PMID: 28376122

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Project description:The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single-molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I-band of a sarcomere is composed of about 40 Ig-domains and is exposed to force under normal physiological conditions and connects the free-hanging ends of the myosin filaments to the Z-disc. Recent single-molecule force spectroscopy data show a mechanical hierarchy in the I-band domains. Domains near the C-terminus in this region unfold at forces two to three times greater than domains near the beginning of the I-band. Though all of these Ig-domains are thought to share a fold and topology common to members of the Ig-like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I-band Ig domain to specific, conserved physical-chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into "strong" and "weak" families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the beta-sandwich fold, and force sensitive residues are not only confined to the A'-G region.

Project description:Rostral laryngeal advancement, also known as laryngeal tie-forward, is used to treat horses for intermittent dorsal displacement of the soft palate and has a morbidity rate of about 6%. We hypothesized that a novel laryngeal clamp would prevent morbidity associated with the sutures tearing through the thyroid cartilage. Larynges (n = 35 horses) were used for ex vivo testing. For uniaxial testing, 15 equine larynges were tested in one of three laryngeal tie-forward constructs [standard laryngeal tie-forward; modified laryngeal tie-forward using a suture-button; and modified laryngeal tie-forward using a laryngeal clamp]. For biaxial testing, 20 larynges were tested in one of two treatment groups: laryngeal tie-forward and laryngeal tie-forward using a laryngeal clamp. Constructs were tested in single cycle-to-failure. Statistical analyses were performed using ANOVA for uniaxial testing and t-tests for biaxial testing. The laryngeal tie-forward using a laryngeal clamp construct was superior to laryngeal tie-forward and laryngeal tie-forward using a suture-button constructs in resistance to pullout in uniaxial testing. The laryngeal tie-forward using a laryngeal clamp presented a significantly different method of failure than the standard laryngeal tie-forward in the biaxial testing. Failure modes for each construct were primarily by suture failure at the clamp (laryngeal tie-forward using a laryngeal clamp), suture pullout through the thyroid cartilage, or, less commonly, tearing of the cricothyroid ligament (laryngeal tie-forward). In uniaxial testing, the laryngeal tie-forward using a laryngeal clamp failed most commonly due to tearing of the cricothyroid ligament, whereas the standard laryngeal tie-forward and the laryngeal tie-forward using a suture-button failed due to the tearing of the cartilage. The laryngeal clamps provided greater stiffness, load at yield, and tensile stress at yield than did the standard construct. Laryngeal clamps may offer an alternative to standard methods of anchoring the thyroid cartilage when performing the laryngeal tie-forward procedure. Further testing and clinical trials are needed to elucidate the utility of the laryngeal tie-forward using a laryngeal clamp.

Dataset Information

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

Publications

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

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