scholarly journals Consensus tetratricopeptide repeat proteins are complex superhelical nanosprings

2021 ◽  
Author(s):  
Marie Synakewicz ◽  
Rohan S. Eapen ◽  
Albert Perez-Riba ◽  
Daniela Bauer ◽  
Andreas Weißl ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Response ◽  
Mechanical Load ◽  
Dynamic Properties ◽  
Cellular Functions ◽  
Tetratricopeptide Repeats ◽  
Ongoing Research ◽  
Repeat Protein ◽  
Repeat Proteins ◽  
Tetratricopeptide Repeat Proteins

AbstractTandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive dynamic properties that are proposed to direct their cellular functions. Here, we report the force response of consensus-designed tetratricopeptide repeats (CTPRs) – superhelical arrays of short helix-turn-helix motifs. Not only are we able to directly observe the repeat-protein superhelix in the force data, but we also find that individual repeats undergo rapid fluctuations between folded and unfolded conformations resulting in a spring-like mechanical response. Using protein engineering, we show how the superhelical geometry can be altered by carefully placed amino-acid substitution and subsequently employ Ising model analysis to examine how these changes affect repeat stability and inter-repeat coupling in ensemble and single-molecule experiments. The Ising models furthermore allow us to map the unfolding pathway of CTPRs under mechanical load revealing how the repeat arrays are unzipped from both ends at the same time. Our findings provide the means to dissect and modulate repeat-protein dynamics and stability, thereby supporting ongoing research efforts into their functional relevance and exploiting them for nanotechnology and biomedical applications.

scholarly journals Regulatory element in fibrin triggers tension-activated transition from catch to slip bonds

2018 ◽  
Vol 115 (34) ◽  
pp. 8575-8580 ◽  
Author(s):  
Rustem I. Litvinov ◽  
Olga Kononova ◽  
Artem Zhmurov ◽  
Kenneth A. Marx ◽  
Valeri Barsegov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Calcium Binding ◽  
Mechanical Response ◽  
Mechanical Stability ◽  
Mechanical Load ◽  
Regulatory Element ◽  
Tensile Force ◽  
Structural Basis ◽  
Bond Rupture ◽  
Calcium Binding Site

Fibrin formation and mechanical stability are essential in thrombosis and hemostasis. To reveal how mechanical load impacts fibrin, we carried out optical trap-based single-molecule forced unbinding experiments. The strength of noncovalent A:a knob-hole bond stabilizing fibrin polymers first increases with tensile force (catch bonds) and then decreases with force when the force exceeds a critical value (slip bonds). To provide the structural basis of catch–slip-bond behavior, we analyzed crystal structures and performed molecular modeling of A:a knob-hole complex. The movable flap (residues γ295 to γ305) containing the weak calcium-binding site γ2 serves as a tension sensor. Flap dissociation from the B domain in the γ-nodule and translocation to knob ‘A’ triggers hole ‘a’ closure, resulting in the increase of binding affinity and prolonged bond lifetimes. The discovery of biphasic kinetics of knob-hole bond rupture is quantitatively explained by using a theory, formulated in terms of structural transitions in the binding pocket between the low-affinity (slip) and high-affinity (catch) states. We provide a general framework to understand the mechanical response of protein pairs capable of tension-induced remodeling of their association interface. Strengthening of the A:a knob-hole bonds at 30- to 40-pN forces might favor formation of nascent fibrin clots subject to hydrodynamic shear in vivo.

scholarly journals Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Albert Perez-Riba ◽  
Elizabeth Komives ◽  
Ewan R. G. Main ◽  
Laura S. Itzhaki
Keyword(s):  
Tandem Repeat ◽  
Modular Architecture ◽  
Tetratricopeptide Repeats ◽  
Repeat Protein ◽  
Repeat Array ◽  
Functional Capabilities ◽  
Repeat Proteins ◽  
Weakly Coupled ◽  
Tandem Repeat Proteins ◽  
Modular Platform

Abstract The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

scholarly journals A Kelch Repeat Protein, Cokel1p, Associates with Microtubules and Is Involved in Appressorium Development in Colletotrichum orbiculare

2010 ◽  
Vol 23 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Ayumu Sakaguchi ◽  
Toshihiko Miyaji ◽  
Gento Tsuji ◽  
Yasuyuki Kubo
Keyword(s):  
Cell Wall ◽  
Wild Type ◽  
Colletotrichum Orbiculare ◽  
Cellular Functions ◽  
Cell Wall Degrading Enzymes ◽  
Calcofluor White ◽  
Repeat Protein ◽  
Repeat Proteins ◽  
Spindle Microtubules ◽  
Appressorium Development

Kelch repeat proteins are conserved in diverse organisms and some are known to mediate fundamental cellular functions. We isolated the gene CoKEL1, encoding a novel kelch repeat protein, from Colletotrichum orbiculare. Analysis of a cokel1 mutant indicated that CoKEL1 is involved in proper appressorium development and cell wall synthesis. Appressoria produced by cokel1 disruption mutants showed irregular shape and impairment of turgor generation and the mutant appressoria rarely penetrated to form infection hyphae in host epidermal cells. Accordingly, cokel1 mutants had reduced pathogenicity on host leaves compared with the wild type. Furthermore, the cokel1 mutant was more sensitive to cell-wall-degrading enzymes and showed altered labeling with the cell wall stain Calcofluor white. Cokel1p was localized on cortical and spindle microtubules in vegetative hyphae. These results suggest that Cokel1p is a microtubule-associated protein involved in infection-related morphogenesis and pathogenicity. This is the first report that a kelch repeat protein is required for the pathogenicity of a fungal plant pathogen.

scholarly journals Folding cooperativity and allosteric function in the tandem-repeat protein class

2018 ◽  
Vol 373 (1749) ◽  
pp. 20170188 ◽  
Author(s):  
Albert Perez-Riba ◽  
Marie Synakewicz ◽  
Laura S. Itzhaki
Keyword(s):  
Binding Site ◽  
Tandem Repeat ◽  
Structural Changes ◽  
Tetratricopeptide Repeats ◽  
Repeat Protein ◽  
Repeat Array ◽  
Repeat Proteins ◽  
Bacterial Quorum Sensing ◽  
Tandem Repeat Proteins ◽  
Bacterial Quorum

The term allostery was originally developed to describe structural changes in one binding site induced by the interaction of a partner molecule with a distant binding site, and it has been studied in depth in the field of enzymology. Here, we discuss the concept of action at a distance in relation to the folding and function of the solenoid class of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats. Distantly located repeats fold cooperatively, even though only nearest-neighbour interactions exist in these proteins. A number of repeat-protein scaffolds have been reported to display allosteric effects, transferred through the repeat array, that enable them to direct the activity of the multi-subunit enzymes within which they reside. We also highlight a recently identified group of tandem-repeat proteins, the RRPNN subclass of TPRs, recent crystal structures of which indicate that they function as allosteric switches to modulate multiple bacterial quorum-sensing mechanisms. We believe that the folding cooperativity of tandem-repeat proteins and the biophysical mechanisms that transform them into allosteric switches are intimately intertwined. This opinion piece aims to combine our understanding of the two areas and develop ideas on their common underlying principles. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

Calibration and Validation of an Elasto-Viscoplastic Material Model for a Hot Work Tool Steel Used in Pressure Casting Dies

2007 ◽  
Vol 345-346 ◽  
pp. 685-688 ◽  
Author(s):  
Werner Ecker ◽  
Thomas Antretter ◽  
R. Ebner
Keyword(s):  
Constitutive Model ◽  
Tool Steel ◽  
Mechanical Response ◽  
Mechanical Load ◽  
Kinematic Hardening ◽  
Ex Situ ◽  
Pressure Casting ◽  
Viscoplastic Material ◽  
Crack Network ◽  
Hot Work Tool Steel

Pressure casting dies are subjected to a large number of thermal as well as mechanical load cycles, which are leading to a characteristic thermally induced crack network on the die surface. As a typical representative for a die material the cyclic thermo-mechanical behavior of the hot work tool steel grade 1.2343 (X38CrMoV5-1) is investigated both experimentally as well as numerically. On the one hand the information from isothermal compression-tension tests is used in a subsequent analysis to calibrate a constitutive model that takes into account the characteristic combined isotropic-kinematic hardening/softening of the material. On the other hand the non-isothermal mechanical response of the material to thermal cycles is characterized by means of a periodic laser pulse applied to a small plate-like specimen which is cooled on the back. The residual stresses developing at the surface of the irradiated region of the specimen are determined ex-situ by means of X-ray diffraction. The obtained values agree well with the results of an accompanying finite-element study. This information is used to verify the calibrated constitutive model. The material law is finally used for the prediction of stresses and strains in a die.

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi

2016 ◽  
Vol 54 (2) ◽  
pp. 136-144 ◽  
Author(s):  
Sunyoung Bang ◽  
Chan-Ki Min ◽  
Na-Young Ha ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim ◽  
...  
Keyword(s):  
Tetratricopeptide Repeat ◽  
Orientia Tsutsugamushi ◽  
Eukaryotic Translation ◽  
Repeat Proteins ◽  
Tetratricopeptide Repeat Proteins

scholarly journals Intrinsic Disorder in Tetratricopeptide Repeat Proteins

2020 ◽  
Vol 21 (10) ◽  
pp. 3709 ◽  
Author(s):  
Nathan W. Van Bibber ◽  
Cornelia Haerle ◽  
Roy Khalife ◽  
Bin Xue ◽  
Vladimir N. Uversky
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Tandem Repeats ◽  
Intrinsically Disordered Protein ◽  
Intrinsic Disorder ◽  
Protein Protein Interactions ◽  
Systematic Analysis ◽  
Tetratricopeptide Repeats ◽  
Intrinsically Disordered ◽  
Tetratricopeptide Repeat Proteins

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.

Sign in / Sign up

Export Citation Format

Share Document