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G M Whitesides

Publications and source records attributed to G M Whitesides.

At least 19 recordsLinked to original sources
PubMed1997

Using protein charge ladders to estimate the effective charges and molecular weights of proteins in solution.

This paper describes the use of capillary electrophoresis (CE) and protein charge ladders to estimate values of effective charge (Z) and molecular weight of proteins under nondenaturing conditions. A panel of 14 proteins with a range of charges and shapes was modified by acetylation with acetic anhydride to yield protein charge ladders. A protein charge ladder is a family of derivatives of a protein that differ in integral units of charge, but minimally in hydrodynamic drag; this mixture of proteins appears in electrophoresis as a set of peaks with regular spacings. Analysis of the electrophoretic mobilities of the members of these charge ladders yields values of Z and electrophoretic coefficients: for a description of mobility based on the equation mu = CPZ(MW)-alpha, CP = 6.3 cm2 min-1 kV-1 charge-1 kD0.48, alpha = 0.48; for mu = C(r)Z[r(1 + kappa r)]-1, C(r) = 55 cm2 min-1 kV-1 charge-1 A (r is the spherical radius of the protein and kappa is a function of ionic strength). The primary usefulness of charge ladders is in measuring the effective charge, Z, of proteins in solution; this information is difficult to obtain by any other procedure. A secondary value of the method is to estimate values of molecular weight. Although less general and convenient than SDS-PAGE, this method allows estimates of molecular weight of nondenatured proteins and is thus applicable to oligomers, noncovalent aggregates, proteins with multiple, non-cross-linked chains, and other systems to which SDS-PAGE is not applicable. The values of molecular weight calculated using the electrophoretic mobilities of proteins in solution and the above constants agreed with literature values to within 20% (with an ambiguous result for ovalbumin). A combination of this technique and SDS-PAGE will be useful in estimating the number of subunits or stage of aggregation of proteins in solution.

Acetic Anhydrides↗
PubMed1996

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Animals↗
PubMed1996

Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.

Amino Acid Sequence↗
PubMed1996

Evaluating electrostatic contributions to binding with the use of protein charge ladders.

Electrostatic interactions between charges on ligands and charges on proteins that are remote from the binding interface can influence the free energy of binding (delta Gb). The binding affinities between charged ligands and the members of a charge ladder of bovine carbonic anhydrase (CAII) constructed by random acetylation of the amino groups on its surface were measured by affinity capillary electrophoresis (ACE). The values of delta Gb derived from this analysis correlated approximately linearly with the charge. Opposite charges on the ligand and the members of the charge ladder of CAII were stabilizing; like charges were destabilizing. The combination of ACE and protein charge ladders provides a tool for quantitatively examining the contributions of electrostatics to free energies of molecular recognition in biology.

Acetylation↗
PubMed1996

A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance.

This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.

Histidine↗
PubMed1996

Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion.

BACKGROUND: The biochemical and biomechanical determinants of adhesion between two biological objects following a collision are complex, and may vary from one system to another. We wished to develop an assay in which all the relevant factors, including the components of the solution, the relative orientation and the relative collision velocity, are under the user's control. RESULTS: A new assay is described in which two mesoscale particles are caused to collide using two independently controlled optical tweezers (optically controlled collision, OPTCOL). This assay enables precise examination of the probability of adhesion under biologically relevant conditions. The OPTCOL assay was used to evaluate the probability of adhesion of a single erythrocyte to a single virus-coated microsphere, in the absence and presence of a sialic acidbearing inhibitor. Inhibition constants for the most effective inhibitors could not be measured using other types of assays. The best inhibitor prevented attachment 50 % of the time at a sialic acid concentration of 35 pmol l-1; it is the most potent known inhibitor of attachment of influenza virus to erythrocytes. CONCLUSIONS: OPTCOL is a versatile new bioassay for studying dynamic interactions in biochemistry. It offers an approach to investigating interactions between moving biological objects that is both quantitative and interpretable. The simplicity of the OPTCOL technique suggests broad applicability to the study of adhesion of mesoscale (1-100 microm) objects in the areas of cell biology, microbiology, medicinal chemistry, and biophysics.

Cell Adhesion↗
PubMed1996

Monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple C-sialoside groups.

BACKGROUND: Influenza viruses use hemagglutinin (HA) arrays to bind to sialic acid moieties on the surface of cells; crosslinking of erythrocytes by this mechanism leads to hemagglutination. A number of synthetic polymers containing multiple sialic acid (Neu5Ac) groups as side chains are potent inhibitors of this process. Inhibition may be due to two mechanisms: polyvalent binding of the inhibitor's multiple Neu5Ac side chains to multiple HA sites on the viral surface, or steric stabilization of the viral particle by a layer of the adsorbed, water-swollen polymer, which prevents adhesion to the erythrocyte. The balance between these two effects is not yet known. RESULTS: Polyacrylamides with multiple C-sialosides (PA(Neu5Ac)) were 2-20 fold more effective as inhibitors of virally mediated hemagglutination when assayed in the presence of Neu2en-NH2, a potent monomeric inhibitor of influenza neuraminidase (NA). The ability of monomeric inhibitors of NA to enhance the inhibition of hemagglutination in this assay correlated with the affinity of the monomer for NA. CONCLUSIONS: We propose that inhibitors of NA act by competing with the C-sialosides of PA(Neu5Ac) for binding to the active sites of the NA. Competitive displacement of Neu5Ac causes an expansion of the layer of polymeric gel absorbed to the virus, enhancing its inhibitory effect. This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.

Acrylic Resins↗
PubMed1996

Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.

Self-assembled monolayers (SAMs) formed on the adsorption of long-chain alkanethiols to the surface of gold or alkylsilanes to hydroxylated surfaces are well-ordered organic surfaces that permit control over the properties of the interface at the molecular scale. The ability to present molecules, peptides, and proteins at the interface make SAMs especially useful for fundamental studies of protein adsorption and cell adhesion. Microcontact printing is a simple technique that can pattern the formation of SAMs in the plane of the monolayer with dimensions on the micron scale. The convenience and broad application offered by SAMs and microcontact printing make this combination of techniques useful for studying a variety of fundamental phenomena in biointerfacial science.

Adsorption↗
PubMed1995

Effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. Insight into mechanism of inhibition.

Highly effective sialic acid-containing inhibitors of influenza virus X-31 were synthesized using poly[N-(acryoyloxy)succinimide] (pNAS), a polymer preactivated by incorporation of active ester groups. Polymers containing two and three different components were prepared by sequential reaction of pNAS with two and three amines, respectively. This preparation of co- and terpolymers was synthetically more efficient than methods involving copolymerization of different monomers and gave polymers that were more easily compared than those generated by copolymerization. Polymers in this study (prepared from a single batch of pNAS) had a constant degree of polymerization (DP approximately 2000) and probably had a distribution of components that was more random than analogous polymers prepared by copolymerization. Use of C-glycosides of sialic acid made it possible to investigate inhibition by different polymers at temperatures ranging from 4 to 36 degrees C without artifacts due to the hydrolytic action of neuraminidase. The inhibitors were, in general, more effective at 36 degrees C than at 4 degrees C. The hemagglutination (HAI) assay was used to measure the value of the inhibition constant KHAIi each polymer. The value of KHAIi for the two-component polymer containing 20% sialic acid on a polyacrylamide backbone at 4 degrees C was 4 nM (in terms of the sialic acid moieties present in solution) and was approximately 50-fold more effective than the best inhibitors previously described and 25-fold more effective than the best naturally occurring inhibitor. The most effective inhibitor synthesized in this work contained 10% benzyl amine and 20% sialic acid on a polyacrylamide backbone, and its value of KHAIi was 600 pM at 36 degrees C. Approximately 100 polymers that differed in one or two components were assayed to distinguish between two limiting mechanisms for inhibition of the interaction between the surfaces of virus and erythrocytes: high-affinity binding through polyvalency, and steric stabilization. The results suggest that both mechanisms play an important role. The system comprising polyvalent inhibitors of agglutination of erythrocytes by influenza provides a system that may be useful as a model for inhibitors of other pathogen-host interactions, a large number of which are themselves polyvalent.

Animals↗
PubMed1995

Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis.

Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG2b antibody and charged ligands that contained a N-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobility of the complex between Ig and its ligand(s). Zwitterionic buffer additives lessened adsorption of protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties and assumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monovalent ligands, the dissociation of the ligands from the antibody occurred noncooperatively. The charge on IgG2b at pH 8.3 is estimated to be -8.0 +/- 0.2; this value is obtained by analysis of the electrophoretic mobilities of complexes IgG2bL2, where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).

Animals↗
PubMed1995

Using capillary electrophoresis to follow the acetylation of the amino groups of insulin and to estimate their basicities.

Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein--insulin and its acylated derivatives--as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G alpha and F alpha) and one epsilon-amino group (K epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (G alpha) = 8.4; pKa (F alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD > or = 6 x 10(3) M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.

Acetylation↗
PubMed1995

Microlithography by using neutral metastable atoms and self-assembled monolayers.

Lithography can be performed with beams of neutral atoms in metastable excited states to pattern self-assembled monolayers (SAMs) of alkanethiolates on gold. An estimated exposure of a SAM of dodecanethiolate (DDT) to 15 to 20 metastable argon atoms per DDT molecule damaged the SAM sufficiently to allow penetration of an aqueous solution of ferricyanide to the surface of the gold. This solution etched the gold and transformed the patterns in the SAMs into structures of gold; these structures had edge resolution of less than 100 nanometers. Regions of SAMs as large as 2 square centimeters were patterned by exposure to a beam of metastable argon atoms. These observations suggest that this system may be useful in new forms of micro- and nanolithography.

Argon↗
PubMed1995

Fabrication of submicrometer features on curved substrates by microcontact printing.

Microcontact printing (mu CP) has been used to produce patterned self-assembled monolayers (SAMs) with submicrometer features on curved substrates with radii of curvature as small as 25 micrometers. Wet-chemical etching that uses the patterned SAMs as resists transfers the patterns formed by mu CP into gold. At present, there is no comparable method for microfabrication on curved surfaces.

Chemistry, Physical↗
PubMed1995

Secondary interactions significantly removed from the sulfonamide binding pocket of carbonic anhydrase II influence inhibitor binding constants.

A series of competitive inhibitors of carbonic anhydrase II (CAII; EC 4.2.1.1) that consists of oligo(ethylene glycol) units attached to p-benzenesulfonamides with pendant amino acids, H2NSO2C6H4CONHCH2CH2OCH2CH2OCH2CH2NHCOCHRNH3+, have been synthesized and examined using competitive fluorescence assays. Three of the strongest inhibitors, designated EG3NH3+, EG3GlyNH3+, and EG3PheNH3+, have been studied by X-ray crystallographic methods at limiting resolutions of 1.9, 2.0, and 2.3 A, respectively. The sulfonamide-zinc binding modes and the association of the ethylene glycol linkers to the hydrophobic patch of the active site are similar in all three inhibitors. Differences in the values of Kd are therefore not due to differences in zinc coordination or to differences in the modes of enzyme-glycol association but instead appear to arise from interaction of the pendant amino acids with the surface of the protein. These pendant groups are, however, not sufficiently ordered to be visible in electron density maps. Thus, structural variations of inhibitors at locations distant from the primary binding (i.e., the sulfonamide group) site affect the overall binding affinities of inhibitors (e.g., Kd (EG3PheNH3+) = 14 nM as compared with Kd (EG3GluNH3+) = 100 nM).

Animals↗
PubMed1995

Increasing binding constants of ligands to carbonic anhydrase by using "greasy tails".

Two series of para-substituted benzenesulfonamides have been examined as inhibitors for bovine carbonic anhydrase II (CAII, EC 4.2.1.1). Both series have hydrophobic alkyl group R connected by amide linkages to the aromatic ring (H2NO2SC6H4-CH2NHCOR1 and H2NO2SC6H4-CONR2R3). The free energy of partitioning (delta Gp) of these ligands between water and octanol had similar, linear correlations with the molecular surface areas of the hydrophobic groups R; delta Gp was only relatively weakly influenced by the linkage to the benzenesulfonamide and the detailed structure of the group R. Binding of these ligands to CAII was more complicated. For compounds having the structure H2NO2SC6H4-L-R, the dependence of the free energy of binding to CAII on the surface area of the hydrocarbon (fluorocarbon) group R for different -L-R was (d delta Gb/dA, kcal/(mol.100 Angstrum 2): -CH2NHCORH, -0.71 +/- 0.03; -CH2NHCORF, -0.72 +/- 0.07; -CONHCH2RH, -2.5 +/- 0.1; and -CONHCH2RF, -2.7 +/- 0.3. The available data permit several conclusions: (i) details (linear, branched, cyclic) of the structure of the group RH are relatively unimportant in determining binding constants (although cyclic structures may bind slightly more strongly than acyclic ligands with the same carbon number); (ii) for a given class of compounds, binding constants of hydrocarbons and fluorocarbons having the same surface area are very similar; and (iii) the nature of the linker L influences the sensitivity of binding to the surface area of the group R, presumably by its influences in positioning the group in the binding pocket of the enzyme. Fluorocarbons seem to be more hydrophobic than hydrocarbons of the same carbon number because they have larger areas of hydrophobic surface; the hydrophobicity of hydrocarbon and fluorocarbon surfaces are similar, after correction for differences in area.

Animals↗
PubMed1994

Determination of the effective charge of a protein in solution by capillary electrophoresis.

This paper describes two methods to estimate the effective charge of a protein in solution by capillary electrophoresis and demonstrates these methods by using representative proteins. In one method, a "charge ladder"--a series of derivatives of a protein differing by known increments of charge but differing only minimally in hydrodynamic drag--is generated by covalent modification of the epsilon-amino groups of lysines with 4-sulfophenyl isothiocyanate or acetic anhydride. In the second method, the equivalent of a charge ladder is produced by noncovalent association of a protein with differently charged ligands. Analysis of the electrophoretic mobilities of the protein and its derivatives as a function of added charge allows the effective charge to be estimated for the unmodified protein. This type of analysis permits estimation of the effective charge of a protein without knowing its composition, structure, or amino acid sequence.

Acetic Anhydrides↗
PubMed1994

Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems.

An alpha-sialoside linked to acrylamide by a short connector (5-acetamido-2-O-(N-acryloyl-8-amino-5-oxaoctyl)-2,6-anhydro-3,5-d ideoxy-D-galacto-alpha-nonulopyranosonoic acid, 1) was prepared. Compound 1 formed high molecular weight copolymers with acrylamide, derivatives of acrylamide, and/or vinylpyrrolidone upon photochemically-initiated free radical polymerization. Those copolymers for which the substituents on the acrylamido nitrogen were small inhibited the agglutination of chicken erythrocytes induced by influenza virus (X-31 (H3N2); a recombinant strain of A/Aichi/2/68 (H3N2) and A/Puerto Rico/8/34 grown in chicken eggs). The inhibitory power of the polymers depended strongly on the conditions of polymerization and the sialic acid content of the polymer. The strongest inhibitors were copolymers (poly(1-co-acrylamide)) formed from mixtures of monomer containing [1]/([1] + [acrylamide]) approximately 0.2-0.7; these copolymers inhibited hemagglutination 10(4)-10(5) times more strongly than did similar concentrations of alpha-methyl sialoside (calculated on the basis of the total concentration of individual sialic acid groups in the solution, whether attached to polymer or present as monomers). Samples polymerized in the presence of low concentrations of cross-linking reagents (bis(acrylamido)methane, BIS, and 2,2'-bis(acrylamido)ethyl disulfide, BAC) also showed increased inhibition (10-10(3)-fold relative to monomers), but their use was limited by their poor solubility. Sterically demanding substituents on any position of the acrylamide component (substituents attached to the vinyl group or N-alkyl groups that are larger than hydroxyethyl) reduced the inhibitory power of the polymer. A 1H NMR assay and a fluorescence depolarization assay showed that poly(1-co-acrylamide) bound to a solubilized trimeric form of the viral receptor for sialic acid (bromelain cleaved hemagglutinin, BHA), less tightly than 1, on a per sialic acid basis. A similar result was also obtained with a model system comprising lactic dehydrogenase (a tetramer) and polymeric derivatives of oxamic acid: that is, poly((28, 29, 30, or 31)-co-acrylamide) had a higher inhibition constant for tetrameric lactic dehydrogenase than did the corresponding monomers (28, 29, 30, or 31) on a per oxamate basis. Poly(1-co-acrylamide) is, in principle, capable of inhibiting the agglutination of erythrocytes by several mechanisms: (1) entropically enhanced binding of the polymer (acting as a polyvalent inhibitor) to the surface of the virus; (2) steric interference of the approach of the virus to the surface of the erythrocyte by a water-swollen layer of the polymer on the surface of the virus; (3) aggregation of the virus induced by the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrylic Resins↗