Construction of protein binding sites in scaffold structures

We have developed a strategy for grafting a protein-protein interface based on the known crystal structure of a native ligand and receptor proteins in a complex. The key interaction residues at the ligand protein binding interface are transferred onto a scaffold protein so that the mutated scaffold protein will bind the receptor protein in the same manner as the ligand protein. First, our method identifies key residues and atoms in the ligand protein, which strongly interact with the receptor protein. Second, this method searches the scaffold protein for combinations of candidate residues, among which the distance between any two candidate residues is similar to that between relevant key interaction residues in the ligand protein. These candidate residues are mutated to key interaction residues in the ligand protein respectively. The scaffold protein is superposed onto the ligand protein based upon the coordinates of corresponding atoms, which are assumed to strongly interact with the receptor protein. Complementarity between scaffold and receptor proteins is evaluated. Scaffold proteins with a low superposing rms difference and high complementary score are accepted for further analysis. Then, the relative position of the scaffold protein is adjusted so that the interfaces between the scaffold and receptor proteins have a reasonable packing density. Other mutations are also considered to reduce the desolvation energy or bad steric contacts. Finally, the scaffold protein is cominimized with the receptor protein and evaluated. To test the method, the binding interface of barstar, the inhibitor of barnase, was grafted onto small proteins. Four scaffold proteins with high complementary scores are accepted.     

Diversity of protein inclusion bodies and identification of mosquitocidal protein in Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis subsp. israelensis produces, during sporulation, protein inclusion bodies of wide ranging sizes, all of which are toxic to mosquitoes. Two proteins are present in the smallest protein bodies (less than 0.2 micron dia.), but the number of proteins increases with increasing size of protein bodies. The largest bodies (greater than 1.5 micron dia.) contain seven proteins. All of the proteins are synthesized at different times during sporulation and are added to developing protein bodies in a stepwise manner. The protein component responsible for mosquitocidal activity is a 65,000-dalton protein, that is present in all of the protein bodies.     

Chloroplast phosphoproteins: phosphorylation of a 12-kDa stromal protein by the redox-controlled kinase of thylakoid membranes

Activation of the redox-controlled protein kinase of thylakoid membranes is detectable in vivo by measuring radioisotope incorporation into the light-harvesting Chl a/b protein and four photosystem II proteins (8.3, 32, 34, and 44 kDa). In normal barley leaves, the kinase is active under both aerobic and anaerobic (N2) conditions, but in the Chl b-less chlorina f2 mutant it is active only under anaerobic conditions. The responsiveness of this enzyme in the mutant to changes in the gas phase has been exploited to distinguish its protein substrates from those of other leaf protein kinases. Most of the soluble phosphoproteins of normal and mutant leaves (including a conspicuously labeled 67-kDa polypeptide) are labeled equally under both aerobic and anaerobic conditions, indicating that they are not substrates of the redox-controlled protein kinase. The major exception is a 12-kDa phosphoprotein, which is labeled in the mutant only under anaerobic conditions. The 67- and 12-kDa phosphoproteins are located in the chloroplast and are labeled when isolated organelles are incubated with [32P]orthophosphate in the light. When thylakoids and stroma are prepared from chloroplasts and are incubated with [gamma-32P]ATP in vitro, the 12-kDa protein is phosphorylated in the thylakoid preparation and then released from the membranes into the medium. The electron transport inhibitor diuron blocks activation of the redox-controlled kinase and prevents phosphorylation of the 12-kDa protein, which is thus the first example of a soluble protein to be phosphorylated by the thylakoid-bound protein kinase. The 67-kDa protein is phosphorylated by a distinct stromal kinase whose activity is not sensitive to diuron.     

Protein Interaction Networks

The study of protein interactions is playing an ever increasing role in our attempts to understand cells and diseases on a system-wide level. This article reviews several experimental approaches that are currently being used to measure protein–protein, protein–DNA and gene–gene interactions. These techniques have now been scaled up to produce extensive genome-wide data sets that are providing us with a first glimpse of global interaction networks. Complementing these experimental approaches, several computational methodologies to predict protein interactions are also reviewed. Existing databases that serve as repositories for protein interaction information and how such databases are used to analyze high-throughput data from a pathway perspective is also addressed. Finally, current efforts to combine multiple data types to obtain more accurate and comprehensive models of protein interactions are discussed. It is clear that the evolution of these experimental and computational approaches is rapidly changing our view of biology, and promises to provide us with an unprecedented ability to model cells and organisms at a system-wide level.     

Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein

Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60-300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini.     

Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli

We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains. The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains. In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains. We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein. Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA. The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature. These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli. Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein.     

Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Protein Thermostability: Mechanism and Control Through Protein Engineering

Chemical modification and protein engineering especially are now the useful tools for thermo-stabilizing proteins, and also for elucidating the mechanism of protein stability. The information on the mechanism so far accumulated indicate that a single or few amino acid replacement(s) in a protein is/are sufficient to enhance protein thermostability. Salt bridges inside protein molecule or decrease of internal or external hydrophobicity, respectively, may contribute to increased thermostability. However, generalized molecular reasons for protein thermostability and generalized methods for protein stabilization have not yet been proposed. Some of typical examples of the application of protein engineering to stabilize proteins are presented. They are based on information concerning the tertiary structure of the proteins or their related proteins. Even if such structural information is unavailable, one can replace amino acid(s) in a protein by mutagenesis of the gene coding for the protein via the application of chemicals to the gene (or the plasmid harbouring the gene) or organism. A promising strategy involving transfer of the identified gene into a thermophile and subsequent growth at higher temperatures (thermal adaptation) is described.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Properties of recA441 protein-catalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein

We have investigated the recombinase activity of recA441 protein by comparing its in vitro DNA strand exchange activity to that of wild-type recA protein. Consistent with its proficiency in recombination in vivo, recA441 protein is able to catalyze the in vitro exchange of a circular single-stranded DNA molecule for a homologous strand in a linear double-stranded DNA molecule. Under conditions optimal for wild-type recA protein, the rates of joint molecule formation are the same for the two recA proteins, but the wild-type protein converts these intermediate species to gapped circular heteroduplex DNA product molecules more rapidly than recA441 protein. In the recA441 protein reaction, joint molecules are instead converted to extensive homology-dependent DNA networks via presumed reinitiation reactions. Under some conditions, the DNA strand exchange activity of recA441 protein is enhanced relative to the wild-type. These conditions include when single-stranded DNA.SSB protein (where SSB is Escherichia coli single-stranded DNA-binding protein) complexes are formed prior to the addition of recA protein, at low magnesium ion concentration in the presence of spermidine, and at low ATP concentrations. Under the conditions examined, recA441 protein competes more effectively with SSB protein for DNA-binding sites; thus, the differences between the strand exchange activities of the wild-type and recA441 proteins can be attributed to this enhanced ability in SSB protein competition.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.     

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis^1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.     

Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data

Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected.     

Evidence of a precursor form of stratum corneum basic protein in rat epidermis.

The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.     

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.     

UniProt archive

UniProt Archive (UniParc) is the most comprehensive, non-redundant protein sequence database available. Its protein sequences are retrieved from predominant, publicly accessible resources. All new and updated protein sequences are collected and loaded daily into UniParc for full coverage. To avoid redundancy, each unique sequence is stored only once with a stable protein identifier, which can be used later in UniParc to identify the same protein in all source databases. When proteins are loaded into the database, database cross-references are created to link them to the origins of the sequences. As a result, performing a sequence search against UniParc is equivalent to performing the same search against all databases cross-referenced by UniParc. UniParc contains only protein sequences and database cross-references; all other information must be retrieved from the source databases.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.