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At least 541 records · Page 30Linked to original sources
PubMed2003

Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties.

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.

Animals↗
PubMed2003

Long-distance conformational changes in a protein engineered by modulated sequence duplication.

There are few, if any, known instances in which a biological signal is transmitted via a large conformational change through the body of a protein. We describe here a mutant of T4 lysozyme that was engineered to permit structural change at a distance. The design uses a tandem sequence repeat that makes it possible to transmit large-scale structural changes from one end of an alpha-helix to the other over a distance of 17-25 A. The method should be of general applicability and may make it possible to introduce a mutation at one site in a protein that will induce large-scale changes in the structure at a spatially remote site.

Binding Sites↗
PubMed1987

Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D9k.

Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.

Aminoquinolines↗
PubMed1987

The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing.

Amino Acid Sequence↗
PubMed2001

Mapping critical sites in collagen II for rational design of gene-engineered proteins for cell-supporting materials.

Collagen II is the most abundant protein of cartilage and forms a network of fibrils extended by proteoglycans that enables cartilage to resist pressure. The surface of the collagen fibril serves as a platform for the attachment of collagen IX, growth factors, and cells. In this study we examined the mechanism of the interaction of chondrocytes with recombinant versions of procollagen II, in which one of the four blocks of 234 amino acids that define repeating D periods of the collagen triple helix has been deleted. Analysis of the attachment of chondrocytes to collagen II variants with deleted D periods indicated that the collagen II monomer contains randomly distributed sites critical for cell binding. However, as was shown by spreading and migration assays, the D4 period, which is between residues 703 to 936, contains amino acids critical for cell motility. We also showed that binding, spreading, and migration of chondrocytes through three-dimensional nanofibrillar collagenous matrices are controlled by an interaction of the collagen triple helix with beta1 integrins. The results of this study provide a basis for the rational design of a scaffold containing genetically engineered collagen with a high density of specific sites of interaction.

Biocompatible Materials↗
PubMed1991

Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen.

Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties. For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors. We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp). In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex. As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold. We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL. By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL. Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors. Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events.

Amino Acid Sequence↗
PubMed1996

Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae.

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximately 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (> 21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Animals↗
PubMed1993

Galloway Memorial Lecture. Protein engineering of tumor necrosis factor-beta and its applications in cancer, septicaemia and cachexia.

The tumour necrosis factors (TNFs) are cytokines, small proteins produced by cells as part of the intercellular signalling network. The exact physiological role of TNFs is unknown, but interest in them focused on three of their capabilities: as potential anti-tumour agents, as humoral mediators of an organism's response to injury and as effector molecules in cachexia. The first TNF to be described, now known as TNF-alpha, has been relatively well studied because the recombinant protein was easily produced since it was cloned in 1984. Studies on TNF-beta or lymphotoxin were hampered by the inability of most groups to express the recombinant protein. This paper describes the expression and purification of recombinant TNF-beta in Escherichia coli, followed by studies to localise the receptor binding site of the molecule through site-directed mutagenesis. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid-50 or tyrosine-108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. This provides evidence for a new hypothesis that both the TNFs bind to their receptors as trimers, each of which is capable of binding simultaneously to three receptors. This leads further to the intriguing possibility of a new mechanism of receptor clustering through simultaneous binding to a single ligand.

Cachexia↗
PubMed1996

A small engineered protein lacks structural uniqueness by increasing the side-chain conformational entropy.

A small globular protein, the third repeat of the c-Myb DNA-binding domain, which is composed of 54 amino acid residues, was engineered so as to understand the structural uniqueness of native proteins. This small protein has three alpha-helices that form a helix-turn-helix structure, which is maintained by the hydrophobic core with three Ile residues. One of the mutant proteins, with two of the buried Ile (Ile-155 and Ile-181) substituted with Leu residues, showed multiple conformations, as monitored by heteronuclear magnetic resonance spectroscopy for 13C- and 15N-labeled proteins. The increase in the side-chain conformational entropy, caused by changing the Ile to a Leu residue on an alpha-helix, could engender the lack of structural uniqueness. In native proteins, the conformations of not only the beta-branched side chains, but also those of the neighboring bulky side chains, can be greatly restricted, depending upon the local backbone structure.

Amino Acid Sequence↗
PubMed1994

Human vascular endothelial cells on expanded PTFE precoated with an engineered protein adhesion factor.

To elucidate the role of the molecular structure of adhesive proteins in an endothelialization of synthetic vascular prosthesis in vitro, a recombinant fibronectin-like engineered adhesion factor (FP) constructed from the specific Arg-Gly-Asp cell adhesion repeats was assayed as adhesive substrate to culture human saphenous vein endothelial cells on ePTFE. ePTFE samples (1 cm2) inserted into cell culture chambers were coated by incubation with increasing amounts of FP (up to 100 micrograms/cm2) prior to cell seeding. At 24 hours after low density cell seeding and 20 micrograms/ml/cm2 FP concentration, the number of adhered cells reached a plateau and the adhered cells did not proliferate up to 6 days of culture. At 24 hours after high density seeding (10(5) cells/cm2), the number of adhered cells was significantly higher on ePTFE with preadsorbed FP than on fibronectin coated PTFE. About 55% of the initially adhered cells survived up to 7 days on FP, whereas cell debris and free nuclei were predominant on fibronectin coated PTFE. In the investigated model the engineered RGD polymer potentialized a short-term adhesion of vascular endothelial cells to PTFE, nevertheless it did not ensure proliferation and long-term survival of these cells.

Blood Vessel Prosthesis↗
PubMed2001

Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids.

The barley high lysine (BHL) proteins are nutritionally enhanced derivatives of barley chymotrypsin inhibitor-2 (CI-2). A compactly folded new CI-2 derivative, BHL9, was engineered with the highest content of threonine, tryptophan, and isoleucine yet achieved in this protein family (15.1, 9.4, and 12.1 wt %, respectively). BHL9 had an unfolding midpoint of 5.5 M guanidinium chloride, significantly greater than values for wild type (3.9 M) or for the previously most stable BHL protein, BHL8 (3.6 M). BHL9 and all other derivatives were digested within 15 s in simulated gastric fluid (SGF), suggesting nutritional availability upon ingestion. Denaturation of the proteins in SGF minus pepsin was revealed by changes in their fluorescence emission spectra and/or far UV circular dichroism spectra. The proteins lack homology to known allergens. Significantly, the BHL8 and BHL9 proteins were stable to proteases at pH 7.5 or 8.0, attesting to their potential for high expression in plants.

Amino Acids, Essential↗
PubMed1988

Monomeric insulins obtained by protein engineering and their medical implications.

The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

Animals↗
PubMed1986

Refined models for computer calculations in protein engineering. Calibration and testing of atomic potential functions compatible with more efficient calculations.

A reappraisal has been made of interatomic potential functions for protein structure calculations using the all-atom approximation (except CH, CH2 and CH3, which are treated as "united atoms"). Some key problems are identified and treated. The potential functions are somewhat novel in form and consistent with more efficient and robust folding algorithms. In addition, the potentials are calibrated for for the rigid geometry approximation, since use of fixed standard bond lengths and valence angles (and fixed trans planar peptide groups) reduces the number of conformational variables and saves a great deal of computer time. Though these algorithms demand the use of potential functions of this special type, these functions can be readily implemented in more classical programs for the conformational analysis of proteins. They are calibrated or tested against a large body of experimental data, including extended basis set ab initio, quantum mechanical calculations, nuclear magnetic resonance spectroscopic data and dipole moment data for di- and oligopeptides, characteristic ratio data for random coil homopolypeptides, extensive data from peptide solubility studies, and experimental structures of polyalanine fibres and globular proteins. This paper will form the basis of a further report, which will include investigations of how water might be more realistically represented subject to the computing power available.

Amino Acid Sequence↗
PubMed1991

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.

Amino Acid Sequence↗