Site-directed Chemical Modification Research Articles

Interrogating conformational dynamics of single-proteins in a plasmonic hotspot

A full understanding of the function of many proteins can only be obtained if different conformations and transitions between them can be monitored in aqueous solution, with adequate temporal resolution. Despite the importance of conformational dynamics, monitoring domain motions of single proteins remains challenging experimentally. Established techniques to determine these conformational changes typically require site-directed chemical modification. The work presented here introduces an approach that traps single unmodified proteins in solution in a plasmonic hotspot and makes it possible to follow changes in protein conformation by concomitant changes in refractive index that can be detected and monitored for minutes to hours with a temporal resolution in the microsecond range.

Quantitative and Site-Directed Chemical Modification of Hypocrellins toward Direct Drug Delivery and Effective Photodynamic Activity

For photodynamic therapy (PDT) treatment of microvascular diseases, drugs are delivered via blood circulation and the targets are vasculature endothelial cells, for which the contradictory requirements of hydrophilicity and lipophilicity of the drugs have been achieved by liposome preparations. Herein, it is demonstrated that the drug delivery and target affinity are achieved by a single chemical compound, hypocrellin B (HB) derivative 6 selected from three novel aminoalkanesulfonic acid HB derivatives, 5-7. 6 exhibits a much higher PDT activity (IC(50) = 22 nM) on human gastric carcinoma BGC823 cells than HB, while it has no cellular toxicity in the dark. On the basis of estimation of the clinically required concentration according to relative PDT activity and clinical criteria, it can be predicted that 6 is directly deliverable to and PDT effective on target cells. The enhanced red absorption and superhigh photoactivity suggest that 6 is more powerful for PDT of tumors than HB.

72 The Choice of Hypoallergens for Fish and Peach to Develop Food Allergy Specific Immunotherapy (TheFAST Project)

BackgroundClassical allergen-specific immunotherapy (SIT), using subcutaneous injections with food extracts, may be effective but dangerous due to anaphylactic side-effects. The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting persistent and severe allergy to fish (cod) and fruit (peach). Both are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. FAST will apply hypo-allergenic recombinant major allergens for SIT.MethodsTwo approaches were evaluated for achieving hypo-allergenicity, i.e. site-directed mutagenesis and chemical modification. Wildtype (wt) natural and recombinant allergens and the hypo-allergens were extensively purified and characterized physico-chemically. Their stability was tested and allergenicity was compared by CAP-inhibition and histamine release experiments while immunogenicity was tested in T-cell proliferation experiments and rabbit and mice immunizations.ResultsFor Cyp c 1, the mutant without calcium-binding site showed up to a 1000 times reduced allergenicity, while secondary fold and immunogenicity (tested in human PBMC stimulations and by immunization of laboratory animals) were retained. Chemically modified Cyp c 1 demonstrated a reduced capacity to stimulate T-cells and showed less immunogenicity in rabbits. The calcium-binding mutant has been produced under GMP conditions.For Pru p 3, 5 potential hypoallergens were compared. The allergenicity was reduced to a similar extent (∼1000-fold) for both variants in which disulfide bridges were disrupted, i.e. either by mutagenesis or by reduction/alkylation. The modification resulted in loss of alpha-helical secondary structure. However, unexpectedly, the immunogenicity was also significantly lowered/absent.ConclusionsFor the Cyp c 1 calcium-binding mutant we are preparing to enter Phase I clinical trials. For Pru p 3, we need to evaluate new molecules to generate a hypoallergenic mutant that retains immunogenicity.

Selective inactivation of M-MuLV RT RNase H activity by site-directed PEGylation: an improved ability to synthesize long cDNA molecules

Moloney murine leukemia virus reverse transcriptase (M-MuLV RT) is a domain structured enzyme that has the N-terminally located DNA polymerization activity and C-terminally located RNase H activity, which interferes with the efficient synthesis of long cDNA molecules. Here we present the PEGylation as a tool for engineering the M-MuLV RT derivative deficient in RNase H activity. We demonstrate that site-directed chemical modification (SDCM) of the RNase H domain by selectively PEGylating C635, one of the eight cysteine residues present in the reverse transcriptase (RT), specifically inactivated its ribonucleolytic activity. As a consequence, the efficiency of long cDNA molecules synthesis by modified enzyme was greatly increased.

Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils

We present site-directed chemical modification (SDCM), a tool for engineering U-resistant archaeal DNA polymerases of family B. The Thermococcus litoralis Sh1B DNA polymerase (GenBank: GQ891548) was chosen as the object of the study. Similar to D.Tok, Kod1, Pfu, Tgo and other archaeal members of this family, the T. litoralis Sh1B DNA polymerase is a domain structured, proofreading-proficient enzyme that has the polymerization and 3′→5′ DNA exonucleolytic activities and contains N-terminally located highly conserved template-strand U-binding pocket. The tight binding of template uracil in the enzyme pocket during polymerization blocks the replication of DNA containing uracils. This effect can be alleviated by mutations in key amino acids of the U-binding pocket. We altered T. litoralis Sh1B DNA polymerase's ability to read through the template-strand uracils by applying SDCM. Specific modification of individual cysteine residues in U-binding pocket — targets introduced into certain positions by site-directed mutagenesis — enables the enzyme to effectively replicate DNA containing uracils. We demonstrate that the acquired resistance of chemically modified T. litoralis Sh1B DNA polymerase to DNA uracil correlates with its decreased affinity for template-strand uracil.

Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers

The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH 2 reduced the activity to 40%. The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase.

Coarse-Grained MD Simulations of Membrane Protein-Bilayer Self-Assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.

“Polar Patch” Proteases as Glycopeptiligases.

The strategy of combined site directed mutagenesis and chemical modification with polar prosthetic groups was used to broaden substrate specificity of proteases resulting in the first successful formation of glycopeptides through the use of glucoamino acid acyl donors in yields of up to 90%.

Alkylation and Cleavage of a DNA Fragment with Trioligonucleotide Reagent

AbstractFor Abstract see ChemInform Abstract in Full Text.

“Polar patch” proteases as glycopeptiligases

The strategy of combined site directed mutagenesis and chemical modification with polar prosthetic groups was used to broaden substrate specificity of proteases resulting in the first successful formation of glycopeptides through the use of glucoamino acid acyl donors in yields of up to 90%.

Efficacy of hypocrellin pharmacokinetics in phototherapy

Hypocrellins A and B are pigments which are isolated from the parasitic fungi Hypocrella bambuase sacc and Shiraia bambusicola P. Heen found in the People's Republic of China and other parts of Asia including Sri Lanka. These agents, which belong to the general class of perylene quinonoid pigments, have a long history of traditional medicinal agents especially in Asia. Hypocrellins are under extensive investigation as photosensitizing agents for photodynamic therapy (PDT). Hypocrellin compounds were selected as potential photosensitizers for PDT owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. The cellular uptake, evaluated by spectrofluorimetry and confocal laser scanning microscopy (CLSM) demonstrated that both HA and HB exhibited high and fast uptake and rapid internalization as revealed by their bio-distribution pattern. In addition, the present study employed both immunocytochemical and Western blot techniques to explore the photo-induced expression of apoptosis related proteins in NPC as well as other human carcinoma cells. Using spectrofluorimetry and CLSM we have determined the cellular fluorescence as a marker for the uptake of HA and HB. Co-staining with either HA or HB and fluorescent dyes specific for cell organelles revealed an intracellular localization of HA and HB in lysosomes other than mitochondria.

Selective reduction and chemical modification of oxidized lipase cysteine mutants

Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.Key words: lipase, cysteine mutant, selective reduction, chemical modification, methanethiosulfonate.

Expanding the utility of proteases in synthesis: broadening the substrate acceptance in non-coded amide bond formation using chemically modified mutants of subtilisin

The strategy of combined site directed mutagenesis and chemical modification creates chemically modified mutants (CMMs) with greatly broadened substrate specificities. We have previously reported that the CMMs of subtilisin Bacillus lentus (SBL) are efficient catalysts for the coupling of both l- and d-amino acids. We now report that these powerful catalysts also allow amide bond formation between a variety of non-coded carboxylic acids, including β-alanine and β-amino homologues of phenylalanine, with both l- and d-amino acid nucleophiles. As a guide to enzyme efficiency, a hydrolysis assay indicating pH change has been employed. CMMs selected by this screen furnished higher yields of coupling products compared to the wild-type enzyme (WT). Furthermore, both WT and CMM enzymes allow highly stereoselective aminolysis of a meso diester with an amino acid amine. These results highlight the utility of CMMs in the efficient formation of non-coded amides as potential peptide isosteres.

Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments.

Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.

Expanded structural and stereospecificity in peptide synthesis with chemically modified mutants of subtilisin

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d-amino acid acyl donors, generating dipeptides containing d-Phe, d-Ala and d-Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l-Ala-NH 2 as acyl acceptors in their S 1′ pockets, which WT-SBL will not.

Site-directed Chemical Modification of Cysteine-scanning Mutants as to Transmembrane Segment II and Its Flanking Regions of the Tn10-encoded Metal-Tetracycline/H+ Antiporter Reveals a Transmembrane Water-filled Channel

Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H+ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580-585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.

Concept of “Binary Oligonucleotide Reagent”

A new strategy for site-directed chemical modification of NA is described. NA-target-driven autoligation reaction between two oligonucleotide derivatives with N-(2-chloroethyl)-N-(p-formylphenyl)-N-propyl-N-3 -ydeneamino and 4-carbohydrazidephenyl groups at their opposing termini results in the NA-target modification, which is several times more effective than modification by one of the derivatives.

Nature of Interaction Between Basic Fibroblast Growth Factor and the Antiangiogenic Drug 7,7-(Carbonyl-bis[imino- N-Methyl-4,2-pyrrolecarbonylimino[ N-methyl-4,2-pyrrole]-carbonylimino])bis-(1,3-naphthalene disulfonate)

PNU145156E (7,7-(carbonyl-bis[imino- N-methyl-4,2-pyrrolecarbonylimino[ N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded K d = 145 nM for a single class of binding sites. Time-resolved anisotropy gave K d = 174 nM. K d = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested.

The CYP2 family: models, mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.

Use of Site-directed Chemical Modification to Study an Essential Lysine in Escherichia coli Leader Peptidase

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.