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PubMed2004

The use of recombinant methods and molecular engineering in protein crystallization.

Recombinant techniques are routinely used for the preparation of protein samples for structural studies including X-ray crystallography. Among other benefits, these methods allow for a vast increase in the amount of obtained protein as compared to purification from source tissues, ease of purification when fusion proteins containing affinity tags are used, introduction of SeMet for phasing, and the opportunity to modify the protein to enhance its crystallizability. Protein engineering may involve removal of flexible regions including termini and interior loops, as well as replacement of residues that affect solubility. Moreover, modification of the protein surface to induce crystal growth may include rational engineering of surface patches that can readily mediate crystal contacts. The latter approach can be used to obtain proteins of crystals recalcitrant to crystallization or to obtain well-diffracting crystals in lieu of wild-type crystals yielding data to limited resolution. This review discusses recent advances in the field and describes a number of examples of diverse protein engineering techniques used in crystallographic investigations.

Chemistry Techniques, Analytical↗
PubMed2001

What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function.

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.

Bacteriophage lambda↗
PubMed2005

Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration.

Model systems mimicking the extracellular matrix (ECM) have greatly helped in quantifying cell migration in three dimensions and elucidated the molecular determinants of cellular motility in morphogenesis, regeneration, and disease progression. Here we tested the suitability of proteolytically degradable synthetic poly(ethylene glycol) (PEG)-based hydrogels as an ECM model system for cell migration research and compared this designer matrix with the two well-established ECM mimetics fibrin and collagen. Three-dimensional migration of dermal fibroblasts was quantified by time-lapse microscopy and automated single-cell tracking. A broadband matrix metalloproteinase (MMP) inhibitor and tumor necrosis factor-alpha, a potent MMP-inducer in fibroblasts, were used to alter MMP regulation. We demonstrate a high sensitivity of migration in synthetic networks to both MMP modulators: inhibition led to an almost complete suppression of migration in PEG hydrogels, whereas MMP upregulation increased the fraction of migrating cells significantly. Conversely, migration in collagen and fibrin proved to be less sensitive to the above MMP modulators, as their fibrillar architecture allowed for MMP-independent migration through preexisting pores. The possibility of molecularly recapitulating key functions of the natural extracellular microenvironment and the improved protease sensitivity makes PEG hydrogels an interesting model system that allows correlation between protease activity and cell migration.

Biocompatible Materials↗
PubMed1994

Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide which shares structural domains with enzymes of the blood clotting cascade. HGF/SF is secreted by cells of mesodermal origin and has powerful mitogenic, motogenic and morphogenic activity on epithelial and endothelial cells. HGF/SF is produced as a biologically inactive single-chain precursor (pro-HGF/SF) most of which is sequestered on the cell surface or bound to the extracellular matrix. Maturation into the active alpha beta heterodimer results from proteolytic cleavage by a urokinase-type protease, which acts as a pro-HGF/SF convertase. The primary determinant for receptor binding appears to be located within the alpha-chain. The interaction of the alpha-chain with the receptor is sufficient for the activation of the signal cascade involved in the motility response. However, the complete HGF/SF protein seems to be required to elicit a mitogenic response. HGF/SF binds with high affinity to a transmembrane receptor, p190MET, encoded by the MET proto-oncogene. p190MET is the prototype of a distinct subfamily of heterodimeric tyrosine kinases, including the putative receptors Ron and Sea. The mature form of p190MET is a heterodimer of two disulfide-linked subunits (alpha and beta). The alpha-subunit is extracellular and heavily glycosylated. The beta-subunit consists of an extracellular portion involved in ligand binding, a membrane spanning segment, and a cytoplasmic tyrosine kinase domain. Both subunits derive from glycosylation and proteolytic cleavage of a common precursor of 170 kDa. In polarized epithelial cells the HGF/SF receptor is selectively exposed in the basolateral plasmalemma, where it is associated with detergent-insoluble components. Two Met isoforms, carrying an intact ligand binding domain but lacking the kinase domain due to truncation of the beta-subunit, arise from alternative post-transcriptional processing of the mature form. One truncated form is soluble and released from the cells. HGF/SF binding triggers tyrosine autophosphorylation of the receptor beta-subunit. Autophosphorylation on the major phosphorylation site Y1235 upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. Negative regulation of the kinase activity occurs through phosphorylation of a unique serine residue (S985) located in the juxtamembrane domain of the receptor. This phosphorylation is triggered by two distinct pathways involving either protein kinase C activation or increase in intracellular Ca2+ concentration. Upon ligand binding, the HGF/SF receptor recruits and activates several cytoplasmic effectors, including phosphatidylinositol 3-kinase (PI 3-K), phospholipase C-gamma (PLC-gamma), pp60c-Src, a tyrosine phosphatase, and a Ras-guanine nucleotide exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2005

Molecular engineering of surfaces using self-assembled monolayers.

The self-assembly of molecules into structurally organized monolayers (SAMs) uses the flexibility of organic chemistry and coordination chemistry to generate well-defined, synthetic surfaces with known molecular and macroscopic properties. The process of designing monolayers with a specified structure gives a high level of control over the molecular-level composition in the direction perpendicular to a surface; soft lithographic technique gives useful (if lower) resolution in the plane of the surface. Alkanethiolates adsorbed on gold, silver, mercury, palladium and platinum are currently the best-defined systems of SAMs. They provide substrates for a number of applications-from studies of wetting and electron transport to patterns for growing mammalian cells. SAMs have made organic surfaces a central part of surface science. Understanding the principles by which they form, and connecting molecular-level structure with macroscopic properties, opens a wide range of areas to study and exploitation.

Adsorption↗
PubMed1997

Molecular engineering with the FRT sequence of the yeast 2 microm plasmid: [cir0] segregant enrichment by counterselection for 2 microm site-specific recombination.

Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.

Cloning, Molecular↗
PubMed1995

Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme.

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.

Amino Acid Sequence↗
PubMed2006

Molecular engineering and measurements to test hypothesized mechanisms in single molecule conductance switching.

Six customized phenylene-ethynylene-based oligomers have been studied for their electronic properties using scanning tunneling microscopy to test hypothesized mechanisms of stochastic conductance switching. Previously suggested mechanisms include functional group reduction, functional group rotation, backbone ring rotation, neighboring molecule interactions, bond fluctuations, and hybridization changes. Here, we test these hypotheses experimentally by varying the molecular designs of the switches; the ability of the molecules to switch via each hypothetical mechanism is selectively engineered into or out of each molecule. We conclude that hybridization changes at the molecule-surface interface are responsible for the switching we observe.

Journal Article↗
PubMed2004

Molecular engineering of myosin.

Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins.

Actins↗
PubMedDate not supplied

Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: biosynthesis and characterization.

The unique advantage of genetic engineering techniques for the design and development of polymers for controlled gene delivery lies in exquisite control over polymer structure. In this article we report the biosynthesis and characterization of a series of new silk-elastinlike protein polymers (SELPs), namely, SELP415K, with larger elastin blocks per monomer unit than SELP47K previously studied for matrix-mediated gene delivery. A new cloning strategy was used, where a block of eight elastin units (8E) was integrated into the existing DNA sequence of SELP47K monomer genes using appropriate restriction endonuclease recognition sites. Following random multimerization, multimer gene segments of desired size were selected, expressed, and purified on Ni-agarose columns. The molecular weight and sequence composition of the purified SELPs were determined by MALDI-TOF and amino acid analysis, respectively. The influence of structural changes on the rheological properties of the polymers was investigated. In addition, hydrogel disks were prepared from 47K and 415K-8mer polymer solutions, and the effects of cure time and environmental conditions on the hydrogel equilibrium swelling ratio as a function of polymer composition were studied. DNA sequencing and agarose gel electrophoresis confirmed the successful cloning of the monomer gene segment of SELP415K consisting of 312 bp. Random concatemerization of SELP415K monomer gene segments resulted in a library of SELP415K multimer sequences of 6, 8, and 10 repeats respectively, each yielding a polymer with exact molecular weight and sequence. Rheometric measurements showed that both complex shear modulus (G*) and gelation point were influenced by polymer composition. Equilibrium swelling studies on hydrogel disks prepared from 47K and 415K-8mer polymer solutions showed that changes in polymer composition resulted in different gelation patterns and increased sensitivity toward changes in temperature and ionic strength but not pH. Together these results demonstrate the potential of recombinant techniques in engineering polymers with defined structures which allows the study of the structural parameters affecting matrix-mediated delivery of genes and bioactive agents.

Amino Acid Sequence↗
PubMed1980

Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions.

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

Base Sequence↗