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PubMed1999

Engineering Novel Functional Proteins Grafting Active Sites into Natural Scaffolds.

Engineering novel small functional proteins by grafting active sites into small but stable proteins is an efficient protein design method. Combining heterogeneous self-consistent ensemble optimization (hetero-SCEO) with 3D-motif search tool, we developed a system to accomplish such method. It is tested by transferring zinc-binding site of carbonic anhydrase form B to charybdotoxin and its efficiency is demonstrated.

Journal Article↗

PubMed1997

Phencyclidine continuous dosing produces a treatment time-dependent regulation of rat CYP2C11 function, protein expression and mRNA levels.

These studies determined the effects of continuous phencyclidine (PCP) administration on cytochrome P450 2C11 (CYP2C11) function, protein expression and mRNA levels. Male Sprague-Dawley rats received s.c. PCP infusions (18 mg/kg/day) for 1, 3, 10 or 20 days (n = 4 per group). Control animals received saline infusions for 3 or 20 days. Livers were collected 24 hr postinfusion, a time when PCP was completely cleared from the animals. In microsomes from the 1- and 3-day PCP infusions, there was a significant decrease (P < .05) in CYP2C11 protein expression (61 and 46% of control values, respectively) and in CYP2C11-mediated metabolism of PCP to a reactive metabolite (36 and 41% of control values). Both protein expression and PCP metabolite formation had returned to normal by 10 days of continuous PCP infusion. CYP2C11 function (as measured by 2alpha-OH testosterone formation) was decreased in the 1-, 3- and 10-day infused rats to 46, 28 and 45% of control values (P < .05). CYP2C11 function, expression and reactive PCP metabolite formation returned to normal after 20 days of PCP infusion. In contrast, CYP2C11 mRNA levels were decreased (P < .05) in liver tissue in PCP-treated rats from 1 to 20 days (43, 31, 37 and 47%, respectively). These data suggest that continuous PCP infusions initially decrease CYP2C11 function and protein expression by a pretranslational mechanism, but continued exposure to PCP leads to metabolic adaptation without the recovery of mRNA levels. Thus, chronic exposure to PCP can produce time-dependent regulation of CYP2C11-mediated metabolism of endogenous and exogenous compounds.

Animals↗

PubMed1987

[Immunologic and functional protein C determination in various internal diseases].

A new practicable and precise functional protein C evaluation test is based on the activation of protein C by a snake venom activator and determination of PC activity by its property to prolong the aPTT in a clotting assay (VK = 1.9% and 4% for intra- and interassay variance respectively). In 40 healthy controls there was a good correlation (r = 0.74) between the functional and immunological (ELISA) evaluation. In 123 patients with both evaluation methods but more pronounced with the measurement of the protein C activity a significant protein C deficiency was found in the patient groups with disseminated solid tumors, inflammatory diseases and myocardial infarction. Besides detection of hereditary PC deficiency Type II (generation of functionally abnormal PC) the functional assay profits by recording PC inhibitor complexes and otherwise dysfunctional PC in DIC. Thus, in patients with hematological neoplasias, only protein C activity was significantly decreases. Decrease of PC activity was more pronounced compared to PC Ag in liver disease indicating synthesis of functionally deficient PC, and in oral anticoagulant treatment due to detection of PIVKA-PC.

Humans↗

PubMed2006

A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Animals↗

PubMed2006

Engineering transport protein function: theoretical and technical considerations using the sugar-transporting phosphotransferase system of Escherichia coli as a model system.

Potential experimental approaches for developing and applying protein-engineering protocols to transmembrane transport systems are described. We specifically consider procedures designed to alter protein function. These procedures are designed for the specific purposes of (1) changing protein interaction specificities and (2) changing a protein's catalytic function. We use sugar-transporting bacterial phosphotransferase systems as model systems to illustrate the proposed approaches. These and other similar procedures are likely to prove to be of utility for biotechnological manipulation of proteins as well as for elucidating potential evolutionary pathways taken for the appearance of novel functions within a protein family.

Bacterial Proteins↗

PubMed2006

Introduction to the membrane protein reviews: the interplay of structure, dynamics, and environment in membrane protein function.

In our review, we introduce an organizational scheme for membrane protein function. It is the relationship between structure, dynamics, and environment that endows the membrane and its constituents with remarkable sensitivity and robustness. Our understanding begins with landmark advances like those presented in the following chapters. Membrane proteins are notoriously difficult to study, and so the work presented here on the ADP/ATP carrier [Nury et al. ( 2 )], rhodopsin [Palczewski ( 24 )], and the cytochrome b6f complex [Cramer et al. ( 35 )] represents incredible progress in this now blossoming field.

Humans↗

PubMed1999

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.

Amino Acid Sequence↗

PubMed1989

Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function.

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.

Amino Acid Sequence↗

PubMed1997

Antigen presentation by MHC class II molecules: invariant chain function, protein trafficking, and the molecular basis of diverse determinant capture.

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4+ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promoters the assembly and transport process, the complex itinerary of class II-invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.

Antigen Presentation↗

PubMed1994

Dominant negative mutants: tools for the study of protein function in vitro and in vivo.

Powerful new approaches for the identification and sequencing of novel cDNAs have produced a backlog of proteins seeking functions. Traditional approaches for characterizing protein function (e.g., blocking monoclonal antibodies and heterologous expression) have significant limitations, especially in identifying the roles specific proteins play in vivo. An alternative approach is to engineer mutations in the protein of interest that abolish its function and that also inhibit the function of simultaneously expressed wild-type protein (dominant negative mutations). This approach has wide application to the study of a number of different kinds of proteins but tends to be most effective for proteins that need to assemble into multimers to be functional. Dominant negative mutants have already provided insights into the molecular mechanisms of action of a number of protein families, including hormone receptors, oncogenes, and growth factor receptors, and have been identified as the cause of at least a few autosomal dominant diseases. Expression of dominant negative mutants under the control of highly active lung cell-specific promoters holds great promise for the study of the roles specific proteins and protein families play in lung development, health, and disease.

Animals↗

PubMed1998

Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases.

The practical exploitation of the vast numbers of sequences in the genome sequence databases is crucially dependent on the ability to identify the function of each sequence. Unfortunately, current methods, including global sequence alignment and local sequence motif identification, are limited by the extent of sequence similarity between sequences of unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence identity. To address this problem, a novel method for identification of protein function based directly on the sequence-to-structure-to-function paradigm is described. Descriptors of protein active sites, termed "fuzzy functional forms" or FFFs, are created based on the geometry and conformation of the active site. By way of illustration, the active sites responsible for the disulfide oxidoreductase activity of the glutaredoxin/thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented. First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the specified activity. Next, these FFFs are used to screen for active sites in low-to-moderate resolution models produced by ab initio folding or threading prediction algorithms. Again, the FFFs can specifically identify the functional sites of these proteins from their predicted structures. The results demonstrate that low-to-moderate resolution models as produced by state-of-the-art tertiary structure prediction algorithms are sufficient to identify protein active sites. Prediction of a novel function for the gamma subunit of a yeast glycosyl transferase and prediction of the function of two hypothetical yeast proteins whose models were produced via threading are presented. This work suggests a means for the large-scale functional screening of genomic sequence databases based on the prediction of structure from sequence, then on the identification of functional active sites in the predicted structure.

Algorithms↗

PubMed1989

Catecholamine-induced heterologous desensitization of rabbit luteal adenylyl cyclase: loss of luteinizing hormone responsiveness is associated with impaired G-protein function.

The effects of injecting epinephrine into pseudopregnant rabbits on the luteal adenylyl cyclase system were analyzed. Epinephrine-induced desensitization was heterologous and associated with a reduced response to isoproterenol, LH, NaF, and forskolin. Epinephrine-induced desensitization was rapid in onset, with a maximum decrease in responsiveness 6 h after treatment and responsiveness returning to control levels within 24 h of treatment. The changes in beta-adrenergic receptor content paralleled changes in catecholamine responsiveness. The affinity of the beta-receptors from treated animals decreased 1.5- to 2-fold before down-regulation. LH receptor number was not altered by epinephrine treatment, although responsiveness to LH was depressed. LH receptor affinity, however, was reduced about 2-fold by epinephrine treatment. Epinephrine treatment also altered G-protein function in corpora lutea, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- membranes and ADP ribosylation by cholera and pertussis toxins. NaF- and isoproterenol-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed for the first 6 h after treatment. The ability of cholera toxin to ADP ribosylate alpha s 46 and alpha s 45 was reduced 1.5-6 h and 3-12 h, respectively, after epinephrine treatment. The reduced ability of cholera toxin to ADP ribosylate alpha s 45 was associated with the decrease in LH receptor affinity after treatment. This supports the contention that alpha s 45 preferentially interacts with the LH receptor. These studies demonstrate that the loss of LH responsiveness upon epinephrine-induced heterologous desensitization is associated with altered G-protein function.

Adenylate Cyclase Toxin↗

PubMed2004

Prediction of Saccharomyces cerevisiae protein functional class from functional domain composition.

MOTIVATION: A key goal of genomics is to assign function to genes, especially for orphan sequences. RESULTS: We compared the clustered functional domains in the SBASE database to each protein sequence using BLASTP. This representation for a protein is a vector, where each of the non-zero entries in the vector indicates a significant match between the sequence of interest and the SBASE domain. The machine learning methods nearest neighbour algorithm (NNA) and support vector machines are used for predicting protein functional classes from this information. We find that the best results are found using the SBASE-A database and the NNA, namely 72% accuracy for 79% coverage. We tested an assigning function based on searching for InterPro sequence motifs and by taking the most significant BLAST match within the dataset. We applied the functional domain composition method to predict the functional class of 2018 currently unclassified yeast open reading frames. AVAILABILITY: A program for the prediction method, that uses NNA called Functional Class Prediction based on Functional Domains (FCPFD) is available and can be obtained by contacting Y.D.Cai at y.cai@umist.ac.uk

Algorithms↗

PubMed1991

Exon organization of the human FKBP-12 gene: correlation with structural and functional protein domains.

FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper we report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting from the topology of the antiparallel beta-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel beta-sheet and alpha-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene also provided insight into the genetic evolution of the immunophilin family. Knowledge of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.

Amino Acid Sequence↗

PubMed2003

Human wild-type SEDL protein functionally complements yeast Trs20p but some naturally occurring SEDL mutants do not.

X-linked spondyloepiphyseal dysplasia tarda (SEDT, or SEDL) is a primary skeletal dysplasia affecting mostly spinal vertebral bodies and epiphyses. Previously, we have identified the SEDL gene and determined the spectrum of 21 different SEDL causing mutations. The SEDL gene is a highly conserved gene with an as yet unknown function. The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex ( approximately 10 proteins) called transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport. While the SEDL gene mutations cause a tissue-specific (epiphyses) and relatively mild phenotype, the Trs20p function is essential for the yeast cell. We now provide evidence that recombinant human SEDL protein is able to functionally complement the Saccharomyces cerevisiae TRS20 (TRAPP subunit 20 gene) knockout mutant. This finding strongly supports the speculated conserved nature of the SEDL/Trs20p function. To shed further light on the SEDL/Trs20p protein function, five different naturally occurring SEDL gene mutations have been tested in complementation studies. While two truncation mutations (157delAT and C271T) and one missense mutation (G139T) were unable to rescue the trs20Delta lethal phenotype, two other missense mutations (C218T and T389A) did complement trs20Delta. Interestingly, there is no obvious correlation between the nature and position of the SEDL mutation and the clinical severity of the disorder among the human SEDL patients. Although the identification of complementing SEDL gene mutations may suggest the existence of subtle phenotypic differences among SEDL patients, it might also point towards the identification of SEDL protein residues/domains specific for normal, vertebrate bone growth.

Amino Acid Sequence↗

PubMed1989

Human chorionic gonadotropin-induced heterologous desensitization of rabbit luteal adenylyl cyclase is associated with altered receptor and G-protein function.

We injected hCG into pseudopregnant rabbits on day 7 of pseudopregnancy and analyzed changes in the components of luteal adenylyl cyclase system in order to determine which components are responsible for altered hormonal responsiveness upon desensitization. hCG-induced desensitization was homologous (loss of responsiveness to LH) early (first 6 h), then became heterologous (partial loss of responsiveness to catecholamines) later (12-48 h). The total number of LH receptors was reduced approximately 30% 3 h after treatment at a time when LH stimulation of adenylyl cyclase activity was not altered. Total LH receptors remained at this level until 24 h, when total receptors were reduced by 88%. While total LH receptor number remained constant, LH-stimulated adenylyl cyclase activity was declining to 57% of the control value at 12 h. Available unoccupied LH receptors were reduced by 96% at 12 h. The affinity of the occupied receptors was reduced 4-fold before down-regulation. The changes in beta-adrenergic receptor number paralleled the changes in catecholamine responsiveness. hCG treatment also altered luteal G-protein function, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- lymphoma membranes and ADP ribosylation by cholera and pertussis toxins. Isoproterenol (ISO)-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed by 65% 12-48 h after hCG treatment, the same time as reduced catecholamine responsiveness. In contrast, NaF-reconstituting activities were at control levels at 12 h and reduced by 55% at 24 and 48 h. Pertussis toxin's ability to ADP ribosylate alpha i 40 was increased 3 and 6 h after treatment, while cholera toxin's ability to ADP ribosylate alpha s 45 was reduced throughout the study period. These studies demonstrate that hCG-induced heterologous desensitization results in a complex series of changes in beta-adrenergic and LH receptors as well as G-protein function, which account for the altered hormonal responsiveness.

Adenylate Cyclase Toxin↗

PubMed2004

Using shared genomic synteny and shared protein functions to enhance the identification of orthologous gene pairs.

MOTIVATION: The identification of orthologous gene pairs is generally based on sequence similarity. Gene pairs that are mutually 'best hits' between the genomes being compared are asserted to be orthologs. Although this method identifies most orthologous gene pairs with high confidence, it will miss a fraction of them, especially genes in duplicated gene families. In addition, the approach depends heavily on the completeness and quality of gene annotation. When the gene sequences are not correctly represented the approach is unlikely to find the correct ortholog. To overcome these limitations, we have developed an approach to identify orthologous gene pairs using shared chromosomal synteny and the annotation of protein function. RESULTS: Assembled mouse and human genomes were used to identify the regions of conserved synteny between these genomes. 'Syntenic anchors' are conserved non-repetitive locations between mouse and human genomes. Using these anchors, we identified blocks of sequences that contain consistently ordered anchors between the two genomes (syntenic blocks). The synteny information has been used to help us identify orthologous gene pairs between mouse and human genomes. The approach combines the mutual selection of the best tBlastX hits between human and mouse transcripts, and inferring gene orthologous relationships based on sharing syntenic anchors, collocating in the same syntenic blocks and sharing the same annotated protein function. Using this approach, we were able to find 19,357 orthologous gene pairs between human and mouse genomes, a 20% increase in the number of orthologs identified by conventional approaches.

Algorithms↗

PubMed2002

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA.

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

Amino Acid Sequence↗