Bacteriorhodopsin protein hybrids for chemical and biological sensing

Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDSPAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the xiv material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse indicating a positive response for targeting of maltose. This chemically linked MBP-bR hybrid protein, with bacteriorhodopsin, as a photoresponsive transducing substrate, shows promise for creation of a universal sensing array by attachment of other pertinent sensing materials, in lieu of the maltose binding protein utilized. This strategy would allow significant reduction in sensor size, while increasing responsiveness and sensitivity at nano and picomolar levels.

Proceedings of the Ninth Annual Biochemical Engineering Symposium

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1979. Since a number of the contributions will be published in detail elsewhere, only brief reports of each contribution are included here. Requests for further information on work at Iowa State University should be directed to Dr. Peter J. Reilly; at Colorado State University to Drs. V. G. Murphy and A. R. Moreira, and at Kansas State University to Drs. L. T. Fan and L. E. Erickson. ContentProperties of a Homogeneous Xylobiohydrolase from Aspergillus niger, Mary M. Frederick, Iowa State University Kinetic Studies on the Enzymatic Hydrolysis of Cellulose–Absorption and Desorption of Cellulase onto Cellulose and the Behavior of Absorbed Cellulase, Yong-Hyun Lee and L. T. Fan, Kansas State University Properties of a Homogeneous Endo-Xylanase from Aspergillus niger, Ricardo Fournier A., Iowa State University Solid State Fermentation of Manure Fibers, D. C. Ulmer, Colorado State University Analysis and Consistency of Experimental Data for Microbial Growth on Renewable Resources, B. 0. Solomon, Kansas State University Biochemical Mechanisms of Enzyme Regulation, Frederick A. Blum, Colorado State University An Evaluation of Cellulose Pretreatments for Enzymatic Hydrolysis, David H. Beardmore, Kansas State University Use of Immobilized 8-Amylase/Glucoamylase Mixtures to Produce High Maltose Syrups, Carol G. Bohnenkamp, Iowa State University Effect of Viscosity on Bubble Behavior in an Airlift Fermentor, Vasanti Deshpande, Kansas State University

PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (Mr 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and β-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thioredoxin and six in-frame histidine residues. The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and β-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities.

A quantitative, high-throughput screen for protein stability

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Using O2 to probe membrane immersion depth by 19F NMR

A fluorinated detergent, CF3(CF2)5C2H4-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by 19F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on 19F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (R1) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O2 at least 100-fold (i.e., PO2 greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (R120bar − R10) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(N,N-dimethyl-N-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in 19F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by 19F NMR.

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal

Previous studies have demonstrated that presecretory proteins such as maltose binding protein (MBP) and outer membrane protein A (OmpA) are targeted to the Escherichia coli inner membrane by the molecular chaperone SecB, but that integral membrane proteins are targeted by the signal recognition particle (SRP). In vitro studies have suggested that trigger factor binds to a sequence near the N terminus of the mature region of OmpA and shunts the protein into the SecB pathway by blocking an interaction between SRP and the signal peptide. By contrast, we have found that the targeting pathway of a protein under physiological conditions is dictated by the composition of its targeting signal. Replacement of the MBP or OmpA signal peptide with the first transmembrane segment of AcrB abolished the dependence on SecB for transport and rerouted both proteins into the SRP targeting pathway. More modest alterations of the MBP signal peptide that simply increase its hydrophobicity also promoted SRP binding. Furthermore, we obtained evidence that SRP has a low affinity for typical signal peptides in vivo. These results imply that different classes of E. coli proteins are targeted by distinct pathways because bacterial SRP binds to a more restricted range of targeting signals than its eukaryotic counterpart.

A method for soluble overexpression of the α7 nicotinic acetylcholine receptor extracellular domain

We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the α7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the α7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist α-bungarotoxin with 2.5 μM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.

Crystal structure of the SarR protein from Staphylococcus aureus

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 Å-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix–turn–helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the “winged helix” family. The dimeric SarR structure could accommodate an unusually long stretch of ≈27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90° or more, similar to that seen in the catabolite activator protein (CAP)–DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

Export of Carbon from Chloroplasts at Night1

Hexose export from chloroplasts at night has been inferred in previous studies of mutant and transgenic plants. We have tested whether hexose export is the normal route of carbon export from chloroplasts at night. We used nuclear magnetic resonance to distinguish glucose (Glc) made from hexose export and Glc made from triose export. Glc synthesized in vitro from fructose-6-phosphate in the presence of deuterium-labeled water had deuterium incorporated at C-2, whereas synthesis from triose phosphates caused C-2 through C-5 to become deuterated. In both tomato (Lycopersicon esculentum L.) and bean (Phaseolus vulgaris L.), Glc from sucrose made at night in the presence of deuterium-enriched water was deuterated only in the C-2 position, indicating that >75% of carbon is exported as hexoses at night. In darkness the phosphate in the cytosol was 28 mm, whereas that in the chloroplasts was 5 mm, but hexose phosphates were 10-fold higher in the cytosol than in the chloroplasts. Therefore, hexose phosphates would not move out of chloroplasts without the input of energy. We conclude that most carbon leaves chloroplasts at night as Glc, maltose, or higher maltodextrins under normal conditions.

A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.