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Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations.

ABSTRACT: The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC2679491 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations.

Zhu Yi Y Bogomolovas Julius J Labeit Siegfried S Granzier Henk H

The Journal of biological chemistry 20090312 20

The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harborin ...[more]

PMID: 19282282

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Project description:Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.

Project description:Multiple interactive domains are involved in the activity of the stress protein, alphaB crystallin that protects against the unfolding, aggregation, and toxicity of amyloidogenic proteins. Six peptides corresponding to the interactive sequences 41STSLSPFYLRPPSFLRAP58, 73DRFSVNLDVKHFS85, 101HGKHEERQDE110, 113FISREFHR120, 131LTITSSLSSDGV142, and 156ERTIPITRE164 in human alphaB crystallin were synthesized and evaluated in Thioflavin T fluorescence assays for their effects on the modulation of fibrillation of four disease-related amyloidogenic proteins: amyloid-beta, alpha-synuclein, transthyretin, and beta2-microglobulin. The 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110 peptides in the conserved alpha crystallin core domain of alphaB crystallin were the most effective fibril inhibitors. 73DRFSVNLDVKHFS85 completely inhibited alpha-synuclein fibrillation and reduced the fibrillation of amyloid-beta, transthyretin, and beta2-microglobulin by >50%. 101HGKHEERQDE110 completely inhibited amyloid-beta fibrillation and reduced the fibrillation of alpha-synuclein, transthyretin, and beta2-microglobulin by >50%. The peptides FSVN, NLDV, HGKH, and HEER, which are synthetic fragments of 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110, inhibited fibrillation of all four amyloidogenic proteins by >75%. In contrast, the peptides FISREFHR, ERTIPITRE, DRFS, KHFS, and EERQ were the strongest promoters of fibrillation. Molecular modeling of the interactions between transthyretin and beta2-microglobulin and the synthetic bioactive peptides determined that residues Phe-75, Ser-76, Val-77, Asn-78, Leu-79, and Asp-80 in 73DRFSVNLDVKHFS85 and residues His-101, Lys-103, His-104, Glu-105, and Arg-107 in 101HGKHEERQDE110 interact with exposed residues in the beta strands, F and D of transthyretin and beta2-microglobulin, respectively, to modulate fibrillation. This is the first characterization of specific bioactive peptides synthesized on the basis of interactive domains in the small heat shock protein, alphaB crystallin that protect against the fibrillation of amyloidogenic proteins.

Project description:Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, betaA3/A1(102-117) peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by beta- and gamma-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of alphaB-crystallin. The present study was performed to identify the interacting sites of the betaA3/A1(102-117) peptide in alphaB-crystallin.betaA3/A1(102-117) peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotin-containing cross-linker. The biotin-derivatized peptide was then incubated with alphaB-crystallin at 37 degrees C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to alphaB-crystallin. Label transfer was confirmed by western blot, and the labeled alphaB-crystallin was digested with trypsin. Tryptic peptides from alphaB-crystallin carrying the biotin label were purified by avidin affinity chromatography, and betaA3/A1(102-117) peptide interacting sites in alphaB-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS).We found that the betaA3/A1(102-117) peptide interacted with alphaB-crystallin regions (70)LEKDR(74), (83)HFSPEELKVK(92), (91)VKVLGDVIEVHGK(103), (93)VLGDVIEVHGKHEER(107), and (121)KYR(123), which are part of the alpha-crystallin domain, and were previously shown to be part of the functional chaperone site in alphaB-crystallin. The betaA3/A1(102-117) peptide also interacted with regions at the COOH-terminal extension of alphaB-crystallin, (150)KQVSGPER(157), (164)EEKPAVTAAPK(174), and (164)EEKPAVTAAPKK(175). When two of the hydrophobic residues of betaA3/A1(102-117) peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property.This study confirmed the interactions between a low molecular weight peptide derived from betaA3/A1-crystallin found in aged and cataract lenses and alphaB-crystallin. The binding of betaA3/A1(102-117) peptide to the chaperone site and the COOH-terminal extension of alphaB-crystallin may explain its anti-chaperone property.

Project description:BackgroundA substitution mutation in human ?A-crystallin (?AG98R) is associated with autosomal dominant cataract. The recombinant mutant ?AG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37 °C. Our previous studies have shown that ?A-crystallin-derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of ?A-crystallin-derived mini-chaperone on the stability and chaperone activity of ?AG98R-crystallin.Methodology/principal findingsRecombinant ?AG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with ?AG98R. The rescuing of chaperone activity in mutant?-crystallin (?AG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of ?AG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized ?AG98R displayed chaperone activity comparable to that of wild-type ?A-crystallin. The complexes formed between mini-?A-?AG98R complex and ADH were more stable than the complexes formed between ?AG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.Conclusion/significanceThese results demonstrate that mini-chaperone stabilizes the mutant ?A-crystallin and modulates the chaperone activity of ?AG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant ?A-crystallins and preserve the chaperone function.

Dataset Information

Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations.

Publications

Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations.

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