Similar Folding Patterns Research Articles

Proteome-scale structural prediction of the giant Marseillevirus reveals conserved folds and putative homologs of the hypothetical proteins.

A significant proportion of the highly divergent and novel proteins of giant viruses are termed "hypothetical" due to the absence of detectable homologous sequences in the existing databases. The quality of genome and proteome annotations often relies on the identification of signature sequences and motifs in order to assign putative functions to the gene products. These annotations serve as the first set of information for researchers to develop workable hypotheses for further experimental research. The structure-function relationship of proteins suggests that proteins with similar functions may also exhibit similar folding patterns. Here, we report the first proteome-wide structure prediction of the giant Marseillevirus. We use AlphaFold-predicted structures and their comparative analysis with the experimental structures in the PDB database to preliminarily annotate the viral proteins. Our work highlights the conservation of structural folds in proteins with highly divergent sequences and reveals potentially paralogous relationships among them. We also provide evidence for gene duplication and fusion as contributing factors to giant viral genome expansion and evolution. With the easily accessible AlphaFold and other advanced bioinformatics tools for high-confidence de novo structure prediction, we propose a combined sequence and predicted-structure-based proteome annotation approach for the initial characterization of novel and complex organisms or viruses.

DSF inactivator RpfB homologous FadD upregulated in Bradyrhizobium japonicum under iron limiting conditions

Phytopathogenic bacteria Xanthomonas campestris pv. campestris (Xcc) causes black rot and other plant diseases. Xcc senses diffusible signal factor (DSF) as a quorum-sensing (QS) signal that mediates mainly iron uptake and virulence. RpfB deactivates DSF in this DSF–QS circuit. We examined differential gene expression profiles of Bradyrhizobium japonicum under low versus high iron conditions and found that fadD and irr were upregulated under low iron (log2 fold change 0.825 and 1.716, respectively). In addition to having similar protein folding patterns and functional domain similarities, FadD shared 58% sequence similarity with RpfB of Xcc. The RpfB–DSF and FadD–DSF complexes had SWISSDock molecular docking scores of − 8.88 kcal/mol and − 9.85 kcal/mol, respectively, and the 100 ns molecular dynamics simulation results were in accord with the docking results. However, significant differences were found between the binding energies of FadD–DSF and RpfB–DSF, indicating possible FadD-dependent DSF turnover. The protein–protein interaction network showed that FadD connected indirectly with ABC transporter permease (ABCtp), which was also upregulated (log2 fold change 5.485). We speculate that the low iron condition may be a mimetic environmental stimulus for fadD upregulation in B. japonicum to deactivate DSF, inhibit iron uptake and virulence of DSF-producing neighbors. This finding provides a new option of using B. japonicum or a genetically improved B. japonicum as a potential biocontrol agent against Xcc, with the added benefit of plant growth-promoting properties.

Brain Folding Patterns May Help Predict Onset of Psychosis

Analysis of MRI scans suggests that differences in cortical gyrification may be able to distinguish at-risk individuals who eventually convert to psychosis from those who do not.

Allele-specific control of replication timing and genome organization during development.

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus × castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment.

Discovering cortical sulcal folding patterns in neonates using large-scale dataset.

The folding of the human cerebral cortex is highly complex and variable across individuals, but certain common major patterns of cortical folding do exist. Mining such common patterns of cortical folding is of great importance in understanding the inter-individual variability of cortical folding and their relationship with cognitive functions and brain disorders. As primary cortical folds are mainly genetically influenced and are well established at term birth, neonates with minimal exposure to the complicated postnatal environmental influences are ideal candidates for mining the major patterns of cortical folding. In this paper, we propose a sulcal-pit-based method to discover the major sulcal patterns of cortical folding. In our method, first, the sulcal pattern is characterized by the spatial distribution of sulcal pits, which are the locally deepest points in cortical sulci. Since deep sulcal pits are genetically related, relatively consistent across individuals, and also stable during brain development, they are well suited for representing and characterizing the sulcal patterns. Then, the similarity between the distributions of sulcal pits is measured from the spatial, geometrical, and topological points of view. Next, a comprehensive similarity matrix is constructed for the whole dataset by adaptively fusing these measurements together, thus capturing both their common and complementary information. Finally, leveraging the similarity matrix, a hierarchical affinity propagation algorithm is used to group similar sulcal folding patterns together. The proposed method has been applied to 677 neonatal brains, and revealed multiple distinct and meaningful sulcal patterns in the central sulcus, superior temporal sulcus, and cingulate sulcus.

Characterization of White Matter Tracts by Diffusion MR Tractography in Cat and Ferret that Have Similar Gyral Patterns.

The developmental relationships between gyral structures and white matter tracts have long been debated, but it is still difficult to discern whether they influence each other's development or are causally related. To explore this topic, this study used cats and ferrets as models for species that share similar gyral folding patterns and imaged with diffusion magnetic resonance imaging to compare white matter innervations in homologous gyri and other brain regions. Adult cat and ferret brains were analyzed via diffusion spectrum imaging tractography and homologous regions of interest were compared. Although similar genetic lineage and gyral structures would suggest analogous white matter tracts, tractography reveals significantly differing white matter connectivity in both the visual and auditory cortices. Similarities in connectivity were concentrated primarily in the highly conserved cerebellar region. These results correlate well with existing histological and functional studies of both species. Our results indicate that, while the 2 species may share similar gyral structures, they utilize different white matter connectivity; suggesting that while species may share similar gyral structures, they can develop different underlying white matter connectivity.

Characterization of a new fungal immunomodulatory protein, FIP-dsq2 from Dichomitus squalens

FIP-dsq2, a new immunomodulatory protein, was identified in Basidiomycota Dichomitus squalens by gene mining. FIP-dsq2 contained 111 amino acids with a molecular weight of 12.51kDa. FIP-dsq2 had a homology range of 51–65% to the reported FIPs. The predicted 3-dimensional model had more similar identical folding patterns in LZ-8 than for FIP-fve. Evolutionary analysis indicated substantial phylogenetic differences were existed with the other FIPs. Overexpression of a 14.07kDa soluble recombinant FIP-dsq2 (rFIP-dsq2) was achieved in Rosetta (pGEX-6P-1) and the purified recombinant protein was homodimer verified by gel filtration chromatography analysis. Antitumour ability of rFIP-dsq2 to human lung adenocarcinoma A549 cells was between LZ-8 and FIP-fve. The cytotoxic effect of rFIP-dsq2 in A549 cancer cells was dose-dependent and the half-maximal inhibitory concentration (IC50) was 15.08μg/mL. Furthermore, rFIP-dsq2 at 8μg/mL could significantly induce apoptosis and interrupt migration in A549 cells. In addition, the antitumour-mechanism exploration suggested that rFIP-dsq2 inhibited A549 proliferation uniquely via apoptotic cell death pathway. The results stated that rFIP-dsq2 was a promising candidate for use in future lung cancer therapy.

Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking

Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.

Leaf-specific pathogenesis-related 10 homolog, PgPR-10.3, shows in silico binding affinity with several biologically important molecules

BackgroundPathogenesis-related 10 (PR-10) proteins are small, cytosolic proteins with a similar three-dimensional structure. Crystal structures for several PR-10 homologs have similar overall folding patterns, with an unusually large internal cavity that is a binding site for biologically important molecules. Although structural information on PR-10 proteins is substantial, understanding of their biological function remains limited. Here, we showed that one of the PgPR-10 homologs, PgPR-10.3, shares binding properties with flavonoids, kinetin, emodin, deoxycholic acid, and ginsenoside Re (1 of the steroid glycosides). MethodsGene expression patterns of PgPR-10.3 were analyzed by quantitative real-time PCR. The three-dimensional structure of PgPR-10 proteins was visualized by homology modeling, and docking to retrieve biologically active molecules was performed using AutoDock4 program. ResultsTranscript levels of PgPR-10.3 expressed in leaves, stems, and roots of 3-wk-old ginseng plantlets were on average 86-fold lower than those of PgPR-10.2. In mature 2-yr-old ginseng plants, the mRNA of PgPR-10.3 is restricted to leaves. Ginsenoside Re production is especially prominent in leaves of Panax ginseng Meyer, and the binding property of PgPR-10.3 with ginsenoside Re suggests that this protein has an important role in the control of secondary metabolism. ConclusionAlthough ginseng PR-10.3 gene is expressed in all organs of 3-wk-old plantlets, its expression is restricted to leaves in mature 2-yr-old ginseng plants. The putative binding property of PgPR-10.3 with Re is intriguing. Further verification of binding affinity with other biologically important molecules in the large hydrophobic cavity of PgPR-10.3 may provide an insight into the biological features of PR-10 proteins.

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca(2+) sensors, which undergo conformational changes in response to oscillating intracellular Ca(2+) concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca(2+)-binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca(2+)-sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca(2+) conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca(2+)-occupied functional state under crowding conditions.

Tryptophan Inhibits Proteus vulgaris TnaC Leader Peptide Elongation, Activating tna Operon Expression

Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP. These residues are conserved in TnaC of P. vulgaris and of other bacterial species. TnaC of P. vulgaris has one additional feature, distinguishing it from TnaC of E. coli; it contains two C-terminal lysine residues following the conserved proline residue. In the present study, we investigated L-tryptophan induction of the P. vulgaris tna operon, transferred on a plasmid into E. coli. Induction was shown to be L-tryptophan dependent; however, the range of induction was less than that observed for the E. coli tna operon. We compared the genetic organization of both operons and predicted similar folding patterns for their respective leader mRNA segments. However, additional analyses revealed that L-tryptophan action in the P. vulgaris tna operon involves inhibition of TnaC elongation, following addition of proline, rather than inhibition of leader peptide termination. Our findings also establish that the conserved residues in TnaC of P. vulgaris are essential for L-tryptophan induction, and for inhibition of peptide elongation. TnaC synthesis is thus an excellent model system for studies of regulation of both peptide termination and peptide elongation, and for studies of ribosome recognition of the features of a nascent peptide.

Two novel defensin-encoding genes of the Chagas disease vector Triatoma brasiliensis (Reduviidae, Triatominae): Gene expression and peptide-structure modeling

Defensins are cysteine-rich peptides involved in the innate immunity of insects and many other organisms. In the present study, two novel defensin-encoding cDNAs and the respective genomic DNAs ( def3 and def4) of Triatoma brasiliensis were identified and their tissue-specific and temporal expression was characterized. Both of the deduced mature peptides consisted of 43 amino acid residues and were highly similar to previously identified triatomine defensins (81.4–100%). Semi-quantitative RT-PCR data showed that def3 was constitutively expressed in the fat body and was induced in salivary glands and the small intestine at 5 and 3 days after feeding (daf), respectively. The def4 mRNA level was highly up-regulated in the stomach and fat-body tissues at 5 and 3 daf, respectively. The three-dimensional structures of these defensins were predicted using a homology modeling approach with Def-AAA, the defensin from Anopheles gambiae, as template (62–74% identity). A map of the electrostatic potential of these models revealed that, despite their similar folding patterns, mature Def2 and Def4 have a more cationic structure than is the case for Def1 and Def3. Such differences may orient the antimicrobial profile of these defensins against distinct targets in different organs of the insect.

Structural search and retrieval using a tableau representation of protein folding patterns

Comparison and classification of folding patterns from a database of protein structures is crucial to understand the principles of protein architecture, evolution and function. Current search methods for proteins with similar folding patterns are slow and computationally intensive. The sharp growth in the number of known protein structures poses severe challenges for methods of structural comparison. There is a need for methods that can search the database of structures accurately and rapidly. We provide several methods to search for similar folding patterns using a concise tableau representation of proteins that encodes the relative geometry of secondary structural elements. Our first approach allows the extraction of identical and very closely-related protein folding patterns in constant-time (per hit). Next, we address the hard computational problem of extraction of maximally-similar subtableaux, when comparing two tableaux. We solve the problem using Quadratic and Linear integer programming formulations and demonstrate their power to identify subtle structural similarities, especially when protein structures significantly diverge. Finally, we describe a rapid and accurate method for comparing a query structure against a database of protein domains, TableauSearch. TableauSearch is rapid enough to search the entire structural database in seconds on a standard desktop computer. Our analysis of TableauSearch on many queries shows that the method is very accurate in identifying similarities of folding patterns, even between distantly related proteins. A web server implementing the TableauSearch is available from http://hollywood.bx.psu.edu/TabSearch.

Functional characterization of Pseudomonas fluorescens OprE and OprQ membrane proteins

Outer membrane (OM) proteins of the OprD family may enable bacteria of the genus Pseudomonas to adapt to various environments by modulating OM permeability. The OprE and OprQ porins from P. fluorescens strain MF0 were purified and identified by MALDI-TOF mass spectrometry and N-terminal and internal microsequencing. These proteins, when reconstituted in an artificial planar lipid bilayer, induced similar ion channels with low single-conductance values. Secondary structure prediction of both proteins showed similar folding patterns into a 16 transmembrane β-strands barrel but a highly variable amino-acid composition and length for their putative external loops implicated in porin function. Both proteins were overexpressed under poor oxygenation conditions, but not by using several amino acids as sole carbon source, indicating a different specificity for these proteins compared to the paradigm of this protein family, OprD.

Similarity and divergence between the RNA polymerase α subunits from hyperthermophilic Thermotoga maritima and mesophilic Escherichia coli bacteria

The α subunit (α Tm) of Thermotoga maritima RNA polymerase has been characterized to investigate its role in transcriptional regulation in one of the few known anaerobic hyperthermophilic bacteria. The highly thermostable α Tm shares 54% similarity with its Escherichia coli analogue (α Ec). The T. maritima rpoA gene coding the α subunit does not complement the thermosensitive rpoA112 mutation of E. coli. However, α Tm and α Ec show similar folding patterns as determined by circular dichroism. Purified α Tm binds to the T. maritima P argGo promoter region (probably to a UP-element) and Arg282 appears to be crucial for DNA binding. The thermostable protein is also able to interact with transcription regulatory proteins, like ArgR from T. neapolitana or CRP from E. coli. These data indicate that the RNA polymerase α subunit might play a crucial role in the modulation of gene expression in hyperthermophiles.

Evolution of Vitamin B 2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.

Zipper-featured delta-peptide foldamers driven by donor--acceptor interaction. Design, synthesis, and characterization.

Donor-acceptor interaction between electron-rich 1,5-dioxynaphthalene (DAN) and electron-deficient pyromellitic diimide (PDI) has been utilized to induce the formation of a new kind of zipper-featured delta-peptide foldamers. Seven l-ornithine-based delta-peptides 1a-g, in which one to three DNA and PDI units are incorporated to the two ends of the peptide backbones, respectively, have been designed and prepared by the standard liquid-phase synthetic method. (1)H NMR, UV-vis, and fluorescent quenching studies reveal that all the delta-peptides adopt folding conformations in nonpolar chloroform and polar DMF as a result of intramolecular donor-acceptor interaction between the DAN and PDI units. The folding states become more compact for the peptide skeletons possessing more donor-acceptor interacting sites. Variable-temperature UV-vis experiments indicate that, although the folding is a dynamic process, the folding state can remain even at 150 degrees C in DMF. Circular dichroism (CD) investigations reveal that the new generation of delta-peptides have similar folding patterns. A zipper-featured folding motif has been proposed for the new generation of delta-peptide foldamers. Molecular modeling has generated two most stable folding states for the longest delta-peptide 1g, with an energy difference of 26.80 kcal/mol.

Diversity among isolates of squash mosaic virus.

cDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87% sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.

The refined crystal structure of Drosophila lebanonensis alcohol dehydrogenase at 1.9 å resolution

Drosophila alcohol dehydrogenase (DADH; EC 1.1.1.1) is a NAD(H)-dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases (SDR) family. This homodimeric enzyme catalyzes the dehydrogenation of alcohols to their respective ketones or aldehydes in the fruit-fly Drosophila, both for metabolic assimilation and detoxification purposes. The crystal structure of the apo form of DADH, one of the first biochemically characterized member of the SDR family, was solved at 1.9 Å resolution by Patterson methods. The initial model was improved by crystallographic refinement accompanied by electron density averaging, R-factor=20.5%, R-free=23.8%.DADH subunits show an α/β single domain structure with a characteristic NAD(H) binding motif (Rossmann fold). The peptide chain of a subunit is folded into a central eight-stranded β-sheet flanked on each side by three α-helices. The dimers have local 2-fold symmetry. Dimer association is dominated by a four-helix bundle motif as well as two C-terminal loops from each subunit, which represent a unique structural feature in SDR enzymes with known structure.Three structural features are characteristic for the active site architecture. (1) A deep cavity which is covered by a flexible loop (33 residues) and the C-terminal tail (11 residues) from the neighboring subunit. The hydrophobic surface of the cavity is likely to increase the specificity of this enzyme towards secondary aliphatic alcohols. (2) The residues of the catalytic triad (Ser138, Tyr151, Lys155) are known to be involved in enzymatic catalysis in the first line. The Tyr151 OH group is involved in an ionic bond with the Lys155 side-chain. Preliminary electrostatic calculations have provided evidence that the active form of Tyr151 is a tyrosinate ion at physiological pH. (3) Three well-ordered water molecules in hydrogen bond distance to side-chains of the catalytic triad may be significant for the proton release steps in DADH catalysis.A ternary structure-based sequence alignment with ten members of the SDR family with known three-dimensional structure has suggested to define a model consisting of four groups of residues, which relates the observed low degree of sequence identity to quite similar folding patterns and nearly identical distributions of residues involved in catalysis.

Peptide Hormones, Steroid Hormones, and Puffs: Mechanisms and Models in Insect Development

Publisher Summary This chapter describes the mechanisms and models of peptide hormones, steroid hormones, and puffs in insect development. The postnatal physical development of homeothermic animals is chiefly a period of increase in size, with a few final touches such as the appearance of secondary sexual characteristics or flight feathers. Perfection of motor and behavioral skills is often the dominant feature of this period. Ecdysteroids act as triggers for alterations in transcription, which, in turn, lead to subsequent cellular and organismal changes. Their action is modified by the juvenile hormone (JH) class of hormones. The classic paradigm of insect endocrinology states that high ecdysteroid titers in the presence of high JH titers lead to larval–larval molts. The cysteines involved in intra- and intermonomer cross-linking of bombyx prothoracicotropic hormone (PTTH) are conserved in the Antheraea and Samia sequences, suggesting very similar folding patterns. The PTTH-stimulated generation of cyclic AMP (cAMP) in prothoracic glands results in rapid activation of a cAMP-dependent protein kinase A (PKA), ∼ 90% of maximum within 5 minutes. The proteins phosphorylated directly by PTTH-activated PKA in the prothoracic glands are not known, it seems likely that PTTH stimulation results in the activation of several additional kinases.