RSS 1.0GBF series
Recent Submissions
-
HUMAN GLYCOPROTEINS AND DERIVED VARIANTS FROM RECOMBINANT MAMMALIAN CELLLINESThe expression of foreign genes using recombinant DNA technology in various host systems has permitted the production of human proteins of therapeutic interest in high amounts. Manyclinically important human proteins are posttranslationally modified. However, the inability of microbes to perform mammalian-type of posttranslational modifications of proteins is a major shortcoming. Alternative expression systems are insect and mammaliancells. Principle mammalian types of protein modifications are N- and O-glycosylation. Insect cells, fungi and yeasts are unable to perform the same terminal glycosylation reactions on glycoproteins as mammalian cells. Recombinant DNA technology used for the production of pharmaceutically useful polypeptides has mainly been focused on microbial expression systems (bacteria like E. coli, yeast and fungi). The advantage of microbial expression systems is the high amount of expressed protein that can be obtained. The present communication considers aspects of glycoprotein research relevant to the field of biotechnology and protein design. Results are presented that have been obtained by our group during the last four years concerning the expression of the glycoproteins human Interleukin 2 (Il-2) and Interferon-8 (IFN-8) in different mammalian cell lines, the determination of their carbohydrate residues, the effect of site-directed mutagenesis on their carbohydrate attachment sites and the insertion of peptide domains which function as acceptors for carbohydrates.
-
COMPUTERAIDED PROTEIN DESIGN: METHODS AND APPLICATIONSSince the first reports on the use ofsite directed mutagenesis in 1982! protein engineering or - when rationally aimed - protein design has been Tecognized as a promising and fascinating field of research in many countries. In Japan (PERI) and the U.S.A. (CARB)researchinstitutes have been founded with the focus on protein design. More and more researchinstitutes in the United States, Canada, Japan and Europe have been Starting broad research projects on protein design (UK: SERC, W. Germany: GBF, EMBL). Possible prospects for applications of designed proteins with modified activities or other new properties are very high,in the areas of pharmacology, enzyme applications in food industry’, waste treatment and chemical synthesis, vaccine design, biosensors etc.*". This conception wasvery clearly lined out in an excellent article by Kevin Ulmerin 19835, Encouraging results have so far been obtained only for a small number ofcases including insulin, proteases and peptidic protease inhibitors, and some others®’. On the other hand many unpredicted andsurprising results of site directed mutagenesis experiments are reported onscientific meetings and in the literature*’, That demonstrates that our methods andtools in thatarea arestill rather crude and urgently require improvement!", Simplecalculations show that the random approach to protein-engineeringis a very slow one. There are 10°* ways to arrange aminoacids in a medium sized protein chain of 250 amino acids. Ca. 10” molecules would form the whole estimated mass ofour universe. But even whenthe information about the seven most important aminoacids is available and only five changes should be tested for each of the positions, about 80,000 different protein-mutants have to be prepared andtested. This implies that a knowledge of the 3D-protein-structure and a good understandingofthe functionactivity relationship is absolutely essential in order to do rational protein-design. Research projects in protein-design require a close cooperation between groupsspecialising in protein-isolation and purification, in fermentation techniques, in genetic-engineering, in DNAsynthesis and protein-crystallography (protein-NMR techniques are being established). This interdisciplinary connection between protein chemists, molecular biologists and stereo-chemists is essential for the protein-design cycle (Fig. 1) consisting of design, cloning, expression andtesting new proteins starting from known ones.
-
NEW DEVELOPMENTSIN PROTEIN CRYSTALLOGRAPHYProtein crystallography is currently undergoing a rapid change in many different ways. One development is the explosion of interest by molecular biologists and immunologists since knowledge of protein sequences, obtained via DNA sequencing, is expanding rapidly, but does often not increase immediately insight into the functioning of the protein. Another change is the recombinant DNA technique which make it possible to obtain large amounts of proteins which were previously only available in minute quantities. A third change is the wide-spread awareness that detailed knowledge of wellselected protein structures is a promising starting point for designing new pharmaceuticals and vaccines, for obtaining new proteins via protein engineering techniques and for inspiring synthetic chemists in their biomimetic endeavours. At the same time many technical aspects of protein crystallography are undergoing a rapid development. Someof them will be describedin this paper. Crystal structures of proteins can be obtained currently in two quite different ways: (i) the "multiple isomorphous replacement" (MIR) method [1-3] for de novo structure determinations, often using additional anomalous scattering information (MIRAS) [4,5]; and, (ii) the "molecular replacement" (MR) method [6-8] for solving new structures related to a known structure. We will discuss the steps involved in obtaining high resolution X-ray structures as outlined in Figure 1. A detailed account of these steps can be found in two volumes of Methods of Enzymology [9].
-
DESIGN AND STRUCTURES OF DISULFIDE CONTAINING SUBTILISIN VARIANTSThe crystal structures of 4 variants of subtilisin, each one containing an engineered disulfide crosslink have been determined. The geometries of the engineered disulfide groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a relationship between their measured redox potentials and their calculated dihedral energies. Disulfide introduction produced cavities in the protein structures. The cavity produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was partially filled by a disordering of nearby Asn117. The cavities were often filled with ordered water molecules that replaced interactions of the removed groups. Molecular modelling provided insight into the location where a disulfide could be incorporated, and into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed that introduction of disulfides into buried hydrophobic regions resulted in long range concerted rearrangements.
-
CHARACTERISATION OF ENGINEERED PROTEINS: SOME CRITICAL REFLECTIONSThis essay is an attempt to point up the gap between, on the one hand, the methods currently available to the biologist in the laboratory and, on the other, the kind of data that he or she would need in order to characterise genetically engineered proteins of topical biological interest in such a way as to make use of the techniques of protein engineering. Sgren Kirkegaard was Denmark's greatest philosopher, and he was well aware of the fact. One day he reflected: “To be Denmark's greatest philosopher, ah, that is indeed a fine satire.” By this he presumably meant that he was the only one. These words have encouraged us to philosophize a little about the protein engineering cycle, of which our version is shownin Figure 1. We have dissected the cycle according to two principles, information-theoretical (vertical axis) and epistemological (horizontal axis). The cycle starts from a gene and proceeds via expression to the corresponding protein, which we associate with a set of properties by testing or suitable characterisation. The understanding of these leads by way of theory, experience or intuition to a new gene, and thereafter the cycle continues, a process of which we have seen many impressive examples.
-
KNOWLEDGE-BASED PROTEIN MODELLING AND DESIGNKnowledge-based modelling can be envisaged as a number of steps concerned with the establishment and use of rules to generate a model of a protein. One of the most powerful procedures in learning rules is comparison of related Structures either through alignment of sequences to identify conserved residues or superposition of three dimensionalstructures to identify conserved conformations or motifs. Thus the first step in a knowledge-based modelling procedure is the systematic comparison of families of topologically similar structures. This step will lead to the establishment of "equivalences" between the structures compared and to their clustering based on measures of similarity. The second step involves the projection of the results of the comparisons of three dimensional structures down onto the level of sequence. This step establishes rules relating sequence to structure. These can be expressed as consensus sequences - templates - for topologically equivalenced residues, or as key residues in canonical structures, which are then used to align the sequence of the protein of unknown tertiary Structure. The third step uses the rules established in the second step to generate a three-dimensional model.
-
DESIGN OF NOVEL INSULINS WITH CHANGED SELF-ASSOCIATION AND LIGAND BINDING PROPERTIESDifferent wild type insulins have been used for treatment of diabetes mellitus since Banting and Best (1) isolated insulin for the first time in 1921. This injection therapy has saved many lives but has not been able to reproduce the serum insulin profile obtained by physiological endogenous insulin secretion. The requirements of biosynthesis, processing and storage in the pancreas have put severe constraints on the insulin molecule giving it properties which are not necessary for the biological action of the hormone and which limit the possibilities of obtaining adequate metabolic control in diabetics.The self-association to a hexamer, for example, facilitates proinsulin conversion and its subsequent precipitation as crystals in the storage vesicle (2) but is evidently not related to the interaction of insulin as a monomer to its receptor, and this property delays the transport of insulin from the subcutaneous depot to the circulation (3). Thus pancreatie insulin did not evolve for exogenous administration, and with the aim of producing insulin with improved therapeutical characteristics a whole series of human insulin analogues with changed self-association and ligand binding properties has been developed through molecular modelling and recombinant DNA-technology. Most analogues were designed to have less tendency to associate (4) but a few were also created to possess stronger association or metal-binding properties.
-
A ComputationalTool for Structural Biology: Crystallographic Refinement by Simulated AnnealingConventional refinementof biological macromolecules involvesa series of steps, each of which consists ofa few cyclesofrestrained least-squares refinement with stereochemical and internal packingconstraints orrestraints that are followed by rebuilding the modelstructure with interactive computer graphics. Duringthefinal stages of refinement solvent molecules are usually included and alternative conformations for some atomsor residues in the protein may be introduced.
-
GENETIC ENGINEERING OF PROTEASE INHIBITORS Alpha1-ANTITRYPSIN AND HIRUDINSite-directed mutagenesis was employed to express different variants of a,antitrypsin in a recombinant strain of Escherichia coli. The first set of variants was designed to render the inhibitor stable under oxidative conditions which reduce the activity of the natural molecule. This was achieved by replacing the methionine residue in position 358 (Pl position) by either valine or leucine. In vitro testing of the variant inhibitors under conditions which mimic the in vivo situation of oxidative stress in the epithelial lining fluid of the lung of cigarette smokers confirmed that they retained their inhibitory efficiency against neutrophil elastase. Furthermore site-directed mutagenesis was used to replace the methionine??® residue with an arginine in order to change the inhibitory specificity of a,antitrypsin from neutrophil elastase to a-thrombin. The design of this inhibitor was based upon the known specificity of a-thrombin for arginine and also upon the presence of an arginine residue in the Pl position of the natural a-thrombin inhibitor antithrombin III. Subsequent in vitro and in vivo evaluation of this variant confirmed that a potent inhibitor of a-thrombin had been designed. Based on further sequence homology studies with other members of the serine protease inhibitor (serpin) family another a@,antitrypsin variant with more pronounced inhibitory effects on plasma kallikrein and factor XIIa was designed. This was achieved by replacing the proline357 residue in the arginine358 variant with an alanine. The double variant matched the Cl-inhibitor in its P2 and P1 positions and proved to be more effective against plasma kallikrein and factor XIIa both in vitro and in vivo than the mutant with only an arginine?>® residue. In a separate set of experiments variants of the naturally occurring a-thrombin inhibitor hirudin were designed and expressed in a recombinant yeast strain. Site-directed mutagenesis experiments established the importance of having a basic residue such as lysine or arginine in position 47 of the inhibitor to obtain efficient thrombin inhibition. In addition it was shown that a replacement of lysine?5 with a threonine residue did not alter the inhibition efficiency thus indicating that the surface loop region around this residue is not involved in the interaction with a-thrombin.
-
THE CROSSOVER LINKER. MECHANISMS AND APPLICATIONS IN GENE MODIFICATIONWe have developed a novel method for mutating DNA sequences, based on site-specific, in vivo, recombination, known as the crossover linker method. A typical crossover linker contains; (i) a single-stranded overhang for an initial cohesive-end ligation with one terminus of a linearized plasmid, (ii) a mid-section carrying modified sequence information, and (iii) a "homology-searching" sequence at the other end, that is similar to a specific region in the opposite terminus of the plasmid. Following transformation of an E. coli host with a plasmid/linker complex, intramolecular recombination between the homologous regions of the resultant intermediate completes the circularization of the plasmid, with concomitant integration of the linker. Crossover linking is performed on double-stranded DNA and can be used to create deletions andinsertions, as well as to perform site-specific mutagenesis. Both single- and double-stranded linkers with "homology searching" region as short as 5 nucleotides can be used for gene modification. Deletions of over 1000 bp have been achieved using "homology searching" regions of approx. 20 nucleotides in length. In this article, the effectiveness, limitations and mechanism of this process are discussed with emphasis on the application of the crossoverlinker to the manipulation of protein-encoding sequences.
-
Computer Simulations Applied to Site Specific Mutagenesis and Ligand Binding: The Use of Free Energy Perturbation MethodsWegive a brief review ofthe theoretical methodology andfree energy perturbation applications to proteins. We show that theoretical methods are capable of complementing experimental studies in leading to a clearer understanding ofthe effect of site specific mutations onligand binding, enzymecatalysis and protein stability.
-
STRUCTURE-FUNCTION RELATIONSHIP IN PARATHYROID HORMONEParathyroid hormone (PTH) is the main regulator of calcium homeostasis and controls resorption as well as formation of bone. We assayed overlapping PTH fragments for their capability to stimulate either adenylate cyclase activity or DNA synthesis in in appropriate renal or skeletal cell systems. From these studies we concluded that PTH-induced enhancement of cellular cAMP levels and of cell proliferation are exerted by different functional domains of the hormone molecule. Their conincidence with structural domains will be discussed. After establishment of an efficient expression system for human PTH in E. coli both functional and structural considerations lead to a series of hormone variants. Their biological characterization showed that individual PTH effects could selectively be affected by these mutations. Therefore, site-directed mutagenesis conceivably opens up the possibility to select for either anabolic or catabolic in vivo activities of PTH. Functional design of a potent inducer-of bone growth might be of considerable clinical interest.
-
STRUCTURAL AND FUNCTIONAL ASPECTS OF PROTEIN-PROTEIN INTERACTION AS STUDIED THROUGH CRYSTAL STRUCTURE OF SUBTILISIN COMPLEXED WITH ITS TRAPPED SUBSTRATE SSI (STREPTOMYCES SUBTILISIN INHIBITOR)The crystal structure of Streptomyces Subtilisin Inhibitor (SSI) was partially refined by restrained least-squares methods to a conventional R value of 24 % employing rotating anode data to 1.85 A resolution range. The Sstructines of the complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitor SSI was partially refined to&® the (sR valuesgot 216% seupkoying lag RR synchrotron data. Comparing the B-factors between free SSI and complexed SSI, the marked rigidification of polypeptide chain segments occurred not only in the “reactive site segment” which is in direct contact with the enzyme but also in those segments which are closely connected with the reactive site segment through either covalent linkage or non-covalent interactions. Moreover the structure of the complex of subtilisin with genetically engineered mutant SSI was solved by ( F mutant - F wild ) difference Fourier syntheses.
-
MODIFICATION OF MILK-CLOTTING ASPARTIC PROTEASES, CHYMOSIN AND MUCOR RENNINArtificial mutagenesis of milk-clotting aspartic proteases, chymosin and a fungal aspartic protease from Mucor pusillus (MPR), was carried out by recombinant DNA techniques. The native and the modified chymosins were prepared by using the expression system of Escherichia coli and their activities were measured with aciddenatured haemoglobin and synthetic peptides. A marked change of substrate specificity with a change of Km or kcat was observed with the mutation of 17077) on’ the flap structure to Phe. Involvement of Lys(221) in determining the pH-activity profile was also suggested. Correctly processed but highly glycosylated MPR was secreted from yeast cells carrying the fungal gene. The decreased milk-clotting activity of the yeast MPR was improved by treatment with endoglycosidase H. Exchange of the Tyr residue on the flap and Trp(45) suggested that the hydrogen bonding between these residues is required for correct arrangement of the S1 subsite. X-ray crystallographic analysis of several aspartic proteases has revealed that 3-dimensional structures of these enzymes are very similar to each other (1). The molecules are bilobal, composing of two topologically similar domains rich in 6 -sheet structures. Their junction forms an extended substrate binding cleft and the two essential aspartyl residues reside at the bottom of the Cleft . A flexible flap region is located at the entrance of the cleft and a tyrosine residue on the flap as well as several hydrophobic residues in the adjacent region are involved to form the Sl subsite for substrate binding. In spite of these high similarity in tertiary structures, marked diversity of the catalytic activity and substrate specificity in these enzymes suggest that different sets of amino acid residues may be involved in their catalytic functions. Calf chymosin and a fungal aspartic protease produced by Mucor pusillus are characterized by their relatively high milk clotting activity and low proteolytic activity, which allow them to be used as milk coagulants in cheese industry. Mutagenesis of these two enzymes was performed by using the recombinant DNA technique to obtain information on the structure-function relationship in this characteristic group of the aspartic proteases.
-
ENGINEERING OF RNASE T 1Ribonuclease Tl (RNase T1) was found in 1975 by Sato and Egamil from Aspergillus oryzae. This enzyme catalyzes hydrolysis of single-stranded RNA at guanylic acid 3'- phosphodiester sites. By this property RNase Tl is extremely useful for elucidation of primary structure of RNA's together with pancreatic RNase. The amino acid sequence of RNase Tl has been reported in 1965 , but it was corrected later in 1985.2 This enzyme consists of 104 amino acids and two disulfide bridges between Cys 2 and 10, as well as Cys6 and 103. RNase T1 is very stable towards heating and acidic conditions and suitable for biochemical and physicochemical studies.3 Recently three dimensional structure of this enzyme was elucidated by X-ray crystallography4:5 as a complex with an inhibitor guanosine 2'-phosphate. In this paper we attempted to obtain some details of structure-function relationship of RNase Tl by means of protein engineering.
-
TERTIARY STRUCTURE OF XYLANASE AND ESTIMATION OF ACTIVE SITES BY SITE DIRECTED MUTAGENESISThe nucleotide sequence of xylanase gene(xynA) of Bacillus pumilus IPO, a hyperproducer of xylanase, was determined and the amino acid sequence was deduced from it. Xylanase is produced as a preenzyme consisting of the mature enzyme of 201 amino acid residues and a signal peptide of 27 residues. Xylanase was analyzed by X-ray crystallography at the level of 2.2 A resolution. It is consisted with two domains, smaller and larger, between two a crevasse suitable to accept xylan molecule was observed. The mutant xylanases obtained by site directed mutagenesis having amino acid alteration; Glu93-Ser93 and Glul82_Asp182 had no catalytic activity (less than 1/10,000).
-
Three Dimensional Structure Determination of Proteins in PERIRibonuclease F,, the guanine-specific ribonuclease from Fusarium moniliforme(1), was crystallized from 2-methyl-2,4- pentanediol/H,O solution in two different crystal forms, corresponding to RNase F,-2'GMP complex or the inhibitor-free enzyme respectively. The molar ratio of 2'GMP/enzyme in the crystals was determined to be 0.9 by comparing absorbances on UV spectra. The inhibitor-free crystal belongs to orthorhombic space group P2,2,2, with unit cell parameters : a=46.6 A, b-56.3 2, c=31.6 A. The crystal of the complex belongs to hexagonal space group P6, with cell dimensions ; a=b=40.2, c=120.9. The inhibitor-free crystal diffracts X-ray very well beyond 1.5 A and intensity data to 1.8 A were collected with a 4 circle diffractometer ( Enraf-Nonius CAD4 ) on a sealed tube generator. Intensity data were also collected from the complex crystal at 2.3 A resolution. RNase F, is by 59 % homologous in sequence with RNase T, (2) of which three dimensional structure was already determined with respect to the 2'GMP-enzyme complex(3,4). The structure analysis of RNase Fy, was carried out about the inhibitor-free crystal, using molecular replacement technique. We could trace the whole main chain of RNase F,. Although its entire conformation including secondary structure is similar to that of RNase Tj, considerable differences were observed in loop structures. This may reflect the conformational alteration caused by binding to 2'GMP (5).
-
TOWARDS THE CONSTRUCTION OF NEW PROTEINSThe construction of new proteins is a challenging goal in present peptide and protein chemistry (2). Success in the chemical synthesis of de novo designed small proteins has been limited until now because of our still limited knowledge of the factors that determine the folding of a polypeptide chain. We have proposed a new strategy which aims at avoiding this problem by the use of the specific possibilities of peptide chemistry, for the synthesis of template— assembled synthetic proteins (TASPs).
-
DESIGN OF EFFICIENT HUMAN LEUKOCYTE ELASTASE INHIBITORS; VARIANTS OF HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR (hPSTI)A set of hPSTI variants were constructed by total gene synthesis or site-specific mutagenesis, with the aim of producing human leukocyte elastase(HLE)-specific inhibitors and obtaining a better insight into the parameters effecting inhibitor/protease interaction. In the initial planning the structure of related Kazal-type inhibitors were taken into account. For subsequent protein design, models were made of HLE based on the structure of porcine pancreatic elastase, and of hPSTI based on the stucture of porcine PSTI. Models of the hPSTI/HLE and hPSTI/chymotrypsin complexes were generated by CAPD "docking". The modelled complexes could be used to rationalise a posteriori the inhibitory properties of the hPSTI variants, and to postulate the structure of better elastase inhibitors which were duly generated. Excellent specific inhibitors (Ky=1.5x107 4" M for SHEE) were obtained and the contribution of individual amino acid residues Or exchanges to the binding constant were estimated.
-
LACTATE DEHYDROGENASE : EFFECT OF AMINO ACID CHANGES ON PROPERTIESMany changes have been made by protein engineering in lactate dehydrogenase from B.stearothermophilus. The role of groups involved in susbtrate and coenzyme binding, catalysis and effector molecule binding has been deduced and variants with improved thermal stability developed. The native substrate catalysis patern of lactate dehydrogenase has been modified by many orders of magnitude, to convert this protein into essentially a highly active malate dehydrogenase. Work is Currently on-going to modify this further and to convert malate dehydrogenase, Similarly by protein engineering, into a functional lactate dehydrogenase.
