scholarly journals Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Edyta Petters ◽  
Daniel Krowarsch ◽  
Jacek Otlewski
Keyword(s):  
Ligand Binding ◽  
Protein Interactions ◽  
Structural Motif ◽  
Tetratricopeptide Repeat ◽  
Protein Protein Interactions ◽  
Denaturation Temperature ◽  
Scanning Calorimetry ◽  
State Process ◽  
Efficient Expression ◽  
Ph Range

Tetratricopeptide repeat (TPR) is a structural motif mediating variety of protein-protein interactions. It has a high potential to serve as a small, stable and robust, non-immunoglobulin ligand binding scaffold. In this study, we showed the consensus approach to design the novel protein called designed tetratricopeptide repeat (dTPR), composed of three repeated 34 amino-acid tetratricopeptide motifs. The designed sequence was efficiently overexpressed in E. coli and purified to homogeneity. Recombinant dTPR is monomeric in solution and preserves its secondary structure within the pH range from 2.0 to 11.0. Its denaturation temperature at pH 7.5 is extremely high (104.5°C) as determined by differential scanning calorimetry. At extreme pH values the protein is still very stable: denaturation temperature is 90.1°C at pH 2.0 and 60.4°C at pH 11. Chemical unfolding of the dTPR is a cooperative, two-state process both at pH 7.5 and 2.0. The free energy of denaturation in the absence of denaturant equals to 15.0 kcal/mol and 13.5 kcal/mol at pH 7.5 and 2.0, respectively. Efficient expression and extraordinary biophysical properties make dTPR a promising framework for a biotechnological application, such as generation of specific ligand- binding molecules.

scholarly journals Roles of the 14-3-3 gene family in cotton flowering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Sang ◽  
Hui Liu ◽  
Bin Ma ◽  
Xianzhong Huang ◽  
Lu Zhuo ◽  
...  
Keyword(s):  
Gene Family ◽  
Protein Interactions ◽  
Leucine Zipper ◽  
Expression Patterns ◽  
Bimolecular Fluorescence Complementation ◽  
Structural Motif ◽  
Virus Induced Gene Silencing ◽  
Protein Protein Interactions ◽  
Basic Leucine Zipper ◽  
Genome Wide

Abstract Background In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown. Results In this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering. Conclusions Here, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

Plasma thermogram profiling: A novel biomarker for lung cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22074-e22074 ◽  
Author(s):  
D. Xiang ◽  
N. C. Garbett ◽  
J. Chaires ◽  
D. L. Laber ◽  
G. H. Kloecker
Keyword(s):  
Lung Cancer ◽  
Healthy Volunteers ◽  
Protein Interactions ◽  
Plasma Proteins ◽  
Molecular Mechanisms ◽  
Plasma Proteome ◽  
Protein Protein Interactions ◽  
Scanning Calorimetry ◽  
Novel Approach ◽  
The Individual

e22074 Background: Currently, there are no clinically useful biomarkers for lung cancer (LC). We developed a novel approach by using differential scanning calorimetry (DSC) to analyze the plasma proteome of LC patients. Our assay produces a signature snapshot of the plasma proteome based on the thermal properties of the constituent proteins within the mixture. Each individual protein has a unique and characteristic melting temperature and melting enthalpy. These biophysical properties are as intrinsic and unique for each protein as are its sequence, mass, and charge. The denaturation of plasma results in a composite signature thermogram that represents the sum of all of the individual proteins within the proteome, weighted according to their concentration within the plasma or serum. Our goal is to identify unique thermogram profiles as biomarkers for lung cancer evaluation. Methods: Plasma samples were collected from LC patients and healthy volunteers. Sample analysis was performed using an automated capillary DSC. Comparison of plasma thermograms from controls and diseased individuals was done using quantile-quantile plots and the Kolmogorov-Smirnov test. Results: One hundred samples were obtained from healthy volunteers. DSC thermograms from control plasma were highly reproducible and yielded a characteristic signature with a well-defined shape and temperature maxima. Twenty samples from LC patients were obtained at diagnosis. Plasma from LC individuals yielded a unique thermogram that differs from that of healthy controls. Preliminary results suggested that DSC was sensitive to properties of the plasma proteins other than their charge and mass. We hypothesize that changes in thermogram shapes and positions in LC can arise from changes in the concentrations of plasma proteins, from disease-induced protein-protein interactions, or from the secretion into plasma of disease-related peptides that can bind to the most abundant plasma proteins. Conclusions: Lung cancer may have a characteristic signature thermogram that may serve as a biomarker for LC; further studies are needed to elucidate the biophysical and molecular mechanisms of different thermograms between normal individuals versus those with disease or cancer. No significant financial relationships to disclose.

Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain

2007 ◽  
Vol 293 (1) ◽  
pp. C95-C105 ◽  
Author(s):  
Ming-Shyue Lee ◽  
I-Chu Tseng ◽  
Youhong Wang ◽  
Ken-ichi Kiyomiya ◽  
Michael D. Johnson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ldl Receptor ◽  
Subcellular Fractionation ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hepatocyte Growth Factor Activator ◽  
Domain 3 ◽  
Ph Range ◽  
Receptor Domain

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 μg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.

scholarly journals Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

2012 ◽  
Vol 448 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jonas Boehringer ◽  
Christiane Riedinger ◽  
Konstantinos Paraskevopoulos ◽  
Eachan O. D. Johnson ◽  
Edward D. Lowe ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Functional Characterization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Structural Motif ◽  
Protein Protein Interactions ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

scholarly journals BB0238, a Presumed Tetratricopeptide Repeat-Containing Protein, Is Required during Borrelia burgdorferi Mammalian Infection

2014 ◽  
Vol 82 (10) ◽  
pp. 4292-4306 ◽  
Author(s):  
Ashley M. Groshong ◽  
Danielle E. Fortune ◽  
Brendan P. Moore ◽  
Horace J. Spencer ◽  
Robert A. Skinner ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Protein Interactions ◽  
Point Mutations ◽  
Structural Motif ◽  
Tetratricopeptide Repeat ◽  
Mammalian Host ◽  
Content Type ◽  
Mammalian Infection ◽  
Tpr Domain

ABSTRACTThe Lyme disease spirochete,Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle ofB. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens.bb0238encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role ofbb0238during mammalian infection, abb0238-deficient mutant was constructed. Thebb0238mutant was attenuated in mice infected via needle inoculation, and complementation ofbb0238expression restored infectivity to wild-type levels.bb0238expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed underin vitroconditions.bb0238is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection.B. burgdorfericlones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in thebb0238deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

scholarly journals Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site

2021 ◽  
Author(s):  
Shiran Lacham-Hartman ◽  
Yulia Shmidov ◽  
Evette S. Radisky ◽  
Ronit Bitton ◽  
David B. Lukatsky ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Protein Interactions ◽  
Protein Complex ◽  
Binding Sites ◽  
Biological Interactions ◽  
Protein Protein Interactions ◽  
Regulatory Pathways ◽  
Multiple Binding Sites ◽  
Ligand Binding Sites

AbstractAlthough myriad protein–protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein–protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.Impact statementLacham-Hartman et al. demonstrate enhancement of binding affinity through avidity in a complex between a bivalent ligand and a soluble monomeric target with a single binding site. Avidity effects have previously been demonstrated only for clustered receptor molecules presenting multiple binding sites. Our model may explain how polyvalent ligands can agonize or antagonize biological interactions involving nonclustered target molecules that are crucial for intra- and extracellular structural, metabolic, signaling, and regulatory pathways.

scholarly journals Chaperone ligand-discrimination by the TPR-domain protein Tah1

2008 ◽  
Vol 413 (2) ◽  
pp. 261-268 ◽  
Author(s):  
Stefan H. Millson ◽  
Cara K. Vaughan ◽  
Chao Zhai ◽  
Maruf M. U. Ali ◽  
Barry Panaretou ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Protein Interactions ◽  
Heat Shock Protein 90 ◽  
Tetratricopeptide Repeat ◽  
Protein Protein Interactions ◽  
Sequence Alignments ◽  
Methionine Residue ◽  
Domain Protein ◽  
Tpr Domain

Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein–protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90α and Hsp90β proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

scholarly journals Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels

2017 ◽  
Vol 292 (43) ◽  
pp. 17718-17730 ◽  
Author(s):  
Kyle A. Lyman ◽  
Ye Han ◽  
Robert J. Heuermann ◽  
Xiangying Cheng ◽  
Jonathan E. Kurz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cyclic Nucleotide ◽  
Tetratricopeptide Repeat ◽  
Hcn Channels ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein–protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)–gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide–binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide–binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization– and co-immunoprecipitation–based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein–protein interactions.

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