The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

The last five amino acid residues at the C-terminus of PRK1 /KKN is essential for full lipid responsiveness

PRK1/PKN is a member of the protein kinase C (PKC) superfamily of serine/threonine protein kinases. Despite its important role as a RhoA effector, limited information is available regarding how this kinase is regulated. We show here that the last seven amino acid residues at the C-terminus is dispensable for the catalytic activity of PRK1 but is critical for the in vivo stability of this kinase. Surprisingly, the intact hydrophobic motif in PRK1 is dispensable for 3-phosphoinositide-dependent kinase-1 (PDK-1) binding and phosphorylation of the activation loop, as the PRK1-Δ940 mutant lacking the last two residues of the hydrophobic motif and the last 5 residues at the C-terminus interacts with PDK-1 in vivo and has a similar specific activity as the wild-type protein. We also found that the last four amino acid residues at the C-terminus of PRK1 is critical for the full lipid responsiveness as the PRK1-Δ942 deletion mutant is no longer activated by arachidonic acid. Our data suggest that the very C-terminus in PRK1 is critically involved in the control of the catalytic activity and activation by lipids. Since this very C-terminal segment is the least conserved among members of the PKC superfamily, it would be a promising target for isozyme-specific pharmaceutical interventions.

Interacting amino acid preferences of 3D pattern pairs at the binding sites of transient and obligate protein complexes

To assess the physico-chemical characteristics of protein-protein interactions, protein sequences and overall structural folds have been analyzed previously. To highlight this, discovery and examination of amino acid patterns at the binding sites defined by structural proximity in 3-dimensional (3D) space are essential. In this paper, we investigate the interacting preferences of 3D pattern pairs discovered separately in transient and obligate protein complexes. These 3D pattern pairs are not necessarily sequence-consecutive, but each residue in two groups of amino acids from two proteins in a complex is within certain °A threshold to most residues in the other group. We develop an algorithm called AA-pairs by which every pair of interacting proteins is represented as a bipartite graph, and it discovers all maximal quasi-bicliques from every bipartite graph to form our 3D pattern pairs. From 112 and 2533 highly conserved 3D pattern pairs discovered in the transient and obligate complexes respectively, we observe that Ala and Leu is the highest occuring amino acid in interacting 3D patterns of transient (20.91%) and obligate (33.82%) complexes respectively. From the study on the dipeptide composition on each side of interacting 3D pattern pairs, dipeptides Ala-Ala and Ala-Leu are popular in 3D patterns of both transient and obligate complexes. The interactions between amino acids with large hydrophobicity difference are present more in the transient than in the obligate complexes. On contrary, in obligate complexes, interactions between hydrophobic residues account for the top 5 most occuring amino acid pairings.

Binding affinities of amino acid analogues at the charged aqueous titania interface : implications for titania-binding peptides

Despite the extensive utilization of biomolecule-titania interfaces, biomolecular recognition and interactions at the aqueous titania interface remain far from being fully understood. Here, atomistic molecular dynamics simulations, in partnership with metadynamics, are used to calculate the free energy of adsorption of different amino acid side chain analogues at the negatively-charged aqueous rutile TiO2 (110) interface, under conditions corresponding with neutral pH. Our calculations predict that charged amino acid analogues have a relatively high affinity to the titania surface, with the arginine analogue predicted to be the strongest binder. Interactions between uncharged amino acid analogues and titania are found to be repulsive or weak at best. All of the residues that bound to the negatively-charged interface show a relatively stronger adsorption compared with the charge-neutral interface, including the negatively-charged analogue. Of the analogues that are found to bind to the titania surface, the rank ordering of the binding affinities is predicted to be "arginine" > "lysine" ≈ aspartic acid > "serine". This is the same ordering as was found previously for the charge-neutral aqueous titania interface. Our results show very good agreement with available experimental data and can provide a baseline for the interpretation of peptide-TiO2 adsorption data.

Changes in the amino acid profiles during embryonic development of the blacklip abalone (Haliotis Rubra)

Changes in the total amino acid (TAA) and the free amino acid (FAA) contents during embryonic development, through newly spawned eggs, to pre-settled larvae of blacklip abalone (Haliotis rubra) are described. The TAA (protein bound + free) and the FAA contents increased prior to hatching but decreased towards settlement, but the changes were not always significant between different stages of development. Threonine, arginine, lysine and leucine accounted for nearly 50 % of the total essential amino acids (TEAA) in all developmental stages. The mean FAA content of newly spawned eggs was 262.8 ± 28.2 pmol·ind^–1 and accounted for 11.5 ± 8.3 % of the TAA. Free essential amino acid (FEAA) content increased significantly as development progressed (P < 0.05), in which threonine, arginine and lysine accounted for over 63 % of this pool. In all developmental stages, the FAA pool was dominated by the non-essential amino acids taurine + proline which accounted for 79.5 % of the total. Generally, the FAA accounted for between 10 to 15 % of the TAA in the different developmental stages of blacklip abalone. All evidence appears to indicate that in blacklip abalone the energy requirements during early ontogeny are mostly met with from the lipid reserves, and that there is a tendency to conserve amino acids until pre-settlement.

Plasma amino acid response after ingestion of different whey protein fractions

Background and objectives The digestion rate of proteins and subsequent absorption of amino acids can independently modulate protein metabolism. The objective of the present study was to examine the blood amino acid response to whey protein isolate (WPI), β-lactoglobulin-enriched WPI, hydrolysed WPI and a flavour-identical control.

Methods Eight healthy adults (four female, four male) were recruited (mean±standard error of the mean: age, 27.0±0.76 years; body mass index, 23.2±0.8 kg/cm2) and after an overnight fast consumed 500 ml of each drink, each containing 25g protein, in a cross-over design. Blood was taken at rest and then every 15 min for 2 h post ingestion.

Results Ingesting the β-lactoglobulin-enriched WPI drink resulted in significantly greater plasma leucine concentrations at 45-120 min and significantly greater branched-chain amino acid concentrations at 60-105 min post ingestion compared with hydrolysed WPI. No differences were observed between WPI and β-lactoglobulin-enriched WPI, and all protein drinks resulted in elevated blood amino acids compared with flavour-identical control.

Conclusions In conclusion, whole proteins resulted in a more rapid absorption of leucine and branched-chain amino acid into the blood compared with the hydrolysed molecular form of whey protein.

The detection of latent fingermarks on porous surfaces using amino acid sensitive reagents : a review

The introduction of ninhydrin treatment as a chemical technique for the visualisation of latent fingermarks on porous surfaces revolutionised approaches to forensic fingermark examination. Since then, a range of amino acid sensitive reagents has been developed and such compounds are in widespread use by law enforcement agencies worldwide. This paper reviews the development and use of these reagents for the detection of latent fingermarks on porous surfaces. A brief overview is provided, including an historical background, forensic significance, and a general approach to the development of latent fingermarks on porous surfaces. This is followed by a discussion of specific amino acid sensitive treatments.

Using polyatomic primary ions to probe an amino acid and a nucleic base in water ice

In this study on pure water ice, we show that protonated water species [H₂O]_nH⁺ are more prevalent than (H₂O)_n⁺ ions after bombardment by Au+ monoatomic and Au₃⁺ and C₆₀⁺ polyatomic projectiles. This data also reveals significant differences in water cluster yields under bombardment by these three projectiles. The amino acid alanine and the nucleic base adenine in solution have been studied and have been shown to have an effect on the water cluster ion yields observed using an Au₃⁺ ion beam.

Solubility and Stability of cytochrome c in hydrated ionic liquids : effect of oxo acid residues and kosmotropicity

Hydrated ionic liquids (ILs) were prepared by adding appropriate amounts of water to hydrophilic ILs. Some hydrated ILs show excellent solubilizing ability for proteins, keeping the basic properties of ILs. The solubility of cytochrome c (cyt c) depended on the structure of the component ions. When component anions have oxo acid residues, the resulting hydrated ILs solubilize cyt c quite well. In such hydrated ILs, the structure and activity of cyt c is influenced by the kosmotropicity of the component ions. We synthesized ILs from various ions having different kosmotropicity, including dihydrogen phosphate (dhp), dibutylphosphate, acetate, lactate, and methanesulfonate as anions. The activity of the dissolved cyt c depends on the permutations of kosmotropicity of the component ions. cyt c shows no structural change and retains its activity when dissolved in the hydrated choline dhp, which is an excellent combination of chaotropic cation and kosmotropic anion. Furthermore, cyt c dissolved in the hydrated choline dhp remained in a native state and was active after 18 months of storage at room temperature.

Substituted naphthoquinones as novel amino acid sensitive reagents for the detection of latent fingermarks on paper surfaces

In this paper, we present our preliminary studies into naphthoquinones as novel reagents for the detection of latent fingermarks on paper. Latent fingermarks deposited on paper substrates were treated with solutions of selected naphthoquinones in ethyl acetate/HFE-7100, with subsequent heating. The selected compounds were 1,4-dihydroxy-2-naphthoic acid, 1,2-naphthoquinone-4-sulfonate, 2-methoxy-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone. All of the tested compounds yielded purple-brown visible fingermarks, which also exhibited photoluminescence when illuminated with a high intensity filtered light source at 555nm and viewed through red goggles. Indirect heat using an oven at 150 ◦C for 1 h was found to be superior to direct heat with an iron, which while providing faster development lead to increased levels of background colouration. Luminescence spectrophotometry revealed differences in photoluminescence characteristics for fingermarks developed with the different naphthoquinones, with excitation over the range 530–590 nm. Luminescence spectrophotometry of developed lysine, glycine and serine spots on paper was used to confirm that the naphthoquinones were reacting with amino acids in the latent fingermark.