Cytokinin Binding Proteins from Phaseolus vulgaris Hypocotyls

t-Zeatin (t-Z) and isopentenyladenosine (iPA) occur naturally as highly active plant cell division regulators, t-Z-Sepherose-4B and iPA-Sepherose-4B affinity column were constructed to isolate and purify the cytokinin-binding proteins from etiolated hypocotyl of Phaseolus vulgaris. Two kinds of cytokinin-binding proteins were obtained. One was 15.5 kD in molecular weight (named ZBP) with only one peptide. The other (named IBP), 165 kD in molecular weight, contained two different subunits (40 kD and 43 kD respectively). The binding activity of ZBP was tested and the dissociation constant (Kd) was determined to be 3.2 × 10-7 mol/L. There was one binding site for t-Z in each molecule of ZBP.     

High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone III

ABSTRACT. Tritiated 10,11-epoxyfarnesyl diazoacetate (EFDA), a photoaffinity label, can be covalently attached to the binding site of a JH-III-specific binding protein in the haemolymph of Locusta migratoria migratorioides (R & F). The specificity of the binding of EFDA to the binding protein is verified by displacement with excess unlabelled JH-III, and EFDA can be used to identify the binding protein in native pore-limiting gradient poly(acrylamide) gel electrophoresis (PAGE) and sodium dodecyl sulphate-PAGE. The native binding protein has a molecular weight of 575,000 and is composed of seemingly identical subunits of molecular weight 81,000.
Three other high-molecular weight serum proteins are identified by native PAGE: a lipophorin, composed of two kinds of apolipophorins, a larval storage protein and a cyanoprotein. The molecular weights and subunit structures of these proteins are investigated, but none of these other high-molecular weight proteins bind JH-III to an appreciable extent.     

A catalytic site of protein disulfide isomerase probed with adenosine-5'-triphosphate analogs

Anthraniloyl adenosine-5'-triphosphate (Ant-ATP) and etheno-adenosine-5'-triphosphate (epsilon-ATP) complexed to Mg(2+) ions are substrates of protein disulfide isomerase (PDI). epsilon-ATP, coordinated to Tb(3+) ions, was used as a probe of the ATPase binding site. Sensitized luminescence arising from resonance energy transfer from epsilon-adenine to Tb(3+) is quenched by PDI. The luminescence results are discussed in reference to a model in which the distance of separation between epsilon-adenine (donor) and Tb(3+) (acceptor) is increased upon binding of PDI. The interaction of a small peptide of 14 amino acid residues with the b/b' domain of the protein does not influence the ATPase activity. The phosphorescence, fluorescence and fluorescence anisotropy of bound epsilon-ATP are not perturbed by the binding of the small molecular weight peptide to PDI. It is suggested that the peptide and ATP do not share a common binding site on the b/b' domain.     

Preparation and characterization of calcium-binding and other hydrophobic proteins from synaptic membranes

A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.5 · 10⁴, a binding capacity of 4 calcium/molecule, and a K_m of 1.5 · 10^?5 M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides.     

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).     

DynaBiS: A hierarchical sampling algorithm to identify flexible binding sites for large ligands and peptides

Knowing the ligand or peptide binding site in proteins is highly important to guide drug discovery, but experimental elucidation of the binding site is difficult. Therefore, various computational approaches have been developed to identify potential binding sites in protein structures. However, protein and ligand flexibility are often neglected in these methods due to efficiency considerations despite the recognition that protein–ligand interactions can be strongly affected by mutual structural adaptations. This is particularly true if the binding site is unknown, as the screening will typically be performed based on an unbound protein structure. Herein we present DynaBiS, a hierarchical sampling algorithm to identify flexible binding sites for a target ligand with explicit consideration of protein and ligand flexibility, inspired by our previously presented flexible docking algorithm DynaDock. DynaBiS applies soft-core potentials between the ligand and the protein, thereby allowing a certain protein–ligand overlap resulting in efficient sampling of conformational adaptation effects. We evaluated DynaBiS and other commonly used binding site identification algorithms against a diverse evaluation set consisting of 26 proteins featuring peptide as well as small ligand binding sites. We show that DynaBiS outperforms the other evaluated methods for the identification of protein binding sites for large and highly flexible ligands such as peptides, both with a holo or apo structure used as input.     

Effects of short-term 17 beta-estradiol treatment on the properties of T4-binding proteins in the plasma of immature rainbow trout, Oncorhynchus mykiss.

To determine the effects of 17 beta-estradiol (E2) on the properties of the plasma proteins that bind L-thyroxine (T4) immature rainbow trout, Oncorhynchus mykiss, were injected intraperitoneally on days 0 and 3 with 0.5 mg E2-3-benzoate/100 g body weight, and plasma was sampled on days 4, 7, or 12. Control trout received peanut oil alone. E2 caused a small but significant decrease in the free T4 index. Saturation analysis on miniature G-25 Sephadex columns revealed at least two major T4-binding sites. Filtration on agarose Bio-gel A 1.5 also indicated two major T4-binding protein fractions with molecular weights of 150 and 55 kDa with a small proportion of T4 binding to a 1,500-kDa site presumed to be lipoprotein. Addition of unlabeled T4 displaced [125I]T4 from the 55-kDa site and unmasked an adjacent site of higher molecular weight. E2 increased the proportion of T4 bound to the low-affinity (150 kDa) site relative to that bound to the high-affinity (55 kDa) site, increased the level of protein associated with the 1,500-kDa site and its T4 binding, and also initiated the production of presumed vitellogenin (VTG), which bound a small amount of T4. It is concluded that the E2-induced depression in FT4 is caused by a shift in T4 binding between high-affinity and low-affinity sites, and also by binding of small amounts of T4 to presumed lipoprotein and VTG.     

The antibiotic thermorubin inhibits protein synthesis by binding to inter-subunit bridge B2a of the ribosome

Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location-the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin's mode of action but possess improved pharmacodynamic properties.     

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.     

Effect of microsphere binding site density on the apparent affinity of an interaction partner.

BACKGROUND: Flow cytometric microsphere-based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner. METHODS: The interaction of a nuclear receptor, peroxisome proliferator-activated receptor gamma ligand binding domain (PPARgamma LBD), with a synthetic peptide derived from a nuclear receptor coactivator protein, PPARgamma coactivator-1 alpha (PGC-1alpha), is the interacting system being studied. The density of this peptide coupled to fluorescently unique microsphere populations is varied by co-incubating the biotinylated peptide and avidin-coated microsphere populations with increasing the amounts of free D-biotin. The discrete-density peptide-coupled microsphere populations are combined to conduct a multiplexed binding experiment with Alexa 532-labeled PPARgamma LBD, in the absence or presence of a small molecule ligand. RESULTS: As the immobilized binding site density of PGC-1alpha peptide on fluorescent microspheres is increased the measured apparent affinity for PPARgamma LBD is increased. CONCLUSIONS: The density of binding sites immobilized to a surface has a pronounced effect on the apparent affinity for soluble binding partners. By controlling and varying the binding site density it is possible to increase the sensitivity of an interaction assay. In multiplexed assay formats it should be possible to normalize intrinsically unequal binding interactions by individually optimizing the binding site density of the immobilized interaction partner. However, to quantitatively measure intrinsic affinities of molecular interactions, low binding site densities are required and multivalent reagents must be avoided.     

Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.     

Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.     

Radicicol-sensitive peptide binding to the N-terminal portion of GRP94

GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on- and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.     

A tetracycline-binding RNA aptamer

Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Nickel binding to the C-terminal tryptic fragment of a peptide from human kidney

In kidney the nickel ion exists primarily as soluble cytoplasmic complexes. We have recently identified a major component of these complexes in the human kidney as a Ni(II) complex of a low molecular weight anionic peptide (Templeton, D.M. and Sarkar, B. (1985) Biochem. J. 230, 35-42). We have now purified a small amount of this peptide to homogeneity and developed an HPLC technique to study its metal-binding properties on sub-nanomole quantities. We are able to demonstrate a binding stoichiometry of one Ni atom per molecule of peptide, with an apparent dissociation constant of 1.1 X 10(-5) M. A similar site exists for Cd. The site for Ni persists after trypsinization, and is localized in the 20-residue C-terminal tryptic fragment of the peptide.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.