PubMed Health⌕ Search

Biomedical subjects

P S Stayton

Publications and source records attributed to P S Stayton.

At least 19 recordsLinked to original sources
PubMed2005

Solid-state NMR structural studies of peptides immobilized on gold nanoparticles.

In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles phi and psi. These angles were determined in turn from distances between backbone carbonyl 13C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of 13C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.

Fourier Analysis↗
PubMed2005

'Smart' delivery systems for biomolecular therapeutics.

OBJECTIVE: There is a strong need for drug delivery systems that can deliver biological signals from biomaterials and tissue engineering scaffolds, and a particular need for new delivery systems that can efficiently deliver biomolecules to intracellular targets. Viruses and pathogens have evolved potent molecular machinery that sense the lowered pH gradient of the endosomal compartment and become activated to destabilize the endosomal membrane, thereby enhancing protein or DNA transport to the cytoplasmic compartment. A key feature of many of these biological delivery systems is that they are reversible, so that the delivery systems are not directly toxic. These delivery systems have the ability to change their structural and functional properties and thus display remarkable 'smart' material properties. The objective of this presentation is to review the initial development of smart polymeric carriers that mimic these biological delivery systems and combine similar pH-sensitive, membrane-destabilizing activity for the delivery of therapeutic biomolecules. DESIGN: We have developed new 'smart' polymeric carriers to more effectively deliver and broaden the available types of biomolecular therapeutics. The polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm. They can be designed to provide a range of pH profiles and membrane-destabilizing activities, allowing their molecular properties to be matched to specific drugs and loading ranges. A versatile set of linker chemistries is available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. RESULTS: The physical properties of several pH-responsive polymers were examined. The activity and pH profile can be manipulated by controlling the length of hydrophobic alkyl segments. The delivery of poly(propyl acrylic acid) (PPAA)-containing lipoplexes significantly enhanced wound healing through the interconnected effects of altered extracellular matrix organization and greater vascularization. PPAA has also been shown to enhance cytoplasmic delivery of a model protein therapeutic. Polymeric carriers displaying pH-sensitive, membrane-destabilizing activity were also examined. The pH profile is controlled by the choice of the alkylacrylic acid monomer and by the ratio of the carboxylate-containing alkylacrylic acid monomer to alkylacrylate monomer. The membrane destabilizing activity is controlled by the lengths of the alkyl segment on the alkylacrylic acid monomer and the alkylacrylate monomer, as well as by their ratio in the final polymer chains. CONCLUSION: The molecular mechanisms that proteins use to sense and destabilize provide interesting paradigms for the development of new polymeric delivery systems that mimic biological strategies for promoting the intracellular delivery of biomolecular drugs. The key feature of these polymers is their ability to directly enhance the intracellular delivery of proteins and DNA, by destabilizing biological membranes in response to vesicular compartment pH changes. The ability to deliver a wide variety of protein and nucleic acid drugs to intracellular compartments from tissue engineering and regenerative scaffolds could greatly enhance control of important processes such as inflammation, angiogenesis, and biomineralization.

Biocompatible Materials↗
PubMed2002

Structural studies of biomaterials using double-quantum solid-state NMR spectroscopy.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composites technologies. Here, we describe both the theory and practice of double-quantum solid-state NMR (ssNMR) structure-determination techniques, as they are used to determine the secondary structures of surface-adsorbed peptides and proteins. In particular, we have used ssNMR dipolar techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Here, we also review NMR data on peptides designed to adsorb from aqueous solutions onto highly porous hydrophobic surfaces with specific helical secondary structures. The adsorption or covalent attachment of biological macromolecules onto polymer materials to improve their biocompatibility has been pursued using a variety of approaches, but key to understanding their efficacy is the verification of the structure and dynamics of the immobilized biomolecules using double-quantum ssNMR spectroscopy.

Amino Acid Sequence↗
PubMed2001

Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.

Amino Acid Sequence↗
PubMed2001

Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield.

Many medical and biotechnological processes rely on controlling and manipulating the molecular-recognition capabilities of proteins. This can be achieved using small molecules capable of competing for protein binding or by changing environmental parameters that affect protein structure and hence binding. An alternative is provided by stimuli-responsive polymers that change reversibly from a water-soluble expanded coil to a water-insoluble collapsed globule upon small changes in temperature, pH or light intensity: when attached to proteins in the vicinity of their binding sites, they reversibly block and release small ligands. Here we show how this approach can be extended to achieve size-selective binding of large, macromolecular ligands. We use the thermally responsive polymer poly(N,N-diethylacrylamide) (PDEAAm), and attach it to the protein streptavidin approximately 20 A from the binding site for biotinylated proteins. Below the lower critical solution temperature of PDEAAm, the polymer is in its extended state and acts as a 'shield' to block the binding of large biotinylated proteins; above this temperature, it collapses and exposes the binding site, thereby allowing binding. We find that the degree of shielding depends on both the size of the biotinylated protein and the size of PDEAAm, suggesting that 'smart' polymer shields could be tailored to achieve a wide range of size-dependent ligand discrimination for use in affinity separations, biosensors and diagnostics technologies.

Acrylamides↗
PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗
PubMed2000

Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion.

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Adsorption↗
PubMed2000

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Antibodies↗
PubMed2000

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system.

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.

Binding Sites↗
PubMed1999

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Binding Sites↗
PubMed1999

Smart and biofunctional streptavidin.

The high affinity recognition of biotin and biotinylated molecules has made streptavidin one of the most important components in diagnostics and laboratory kits. While it is extremely useful as the native protein, there are many applications where its function can be improved re-engineering the subunits. We review here our efforts to construct streptavidin tetramers that have 'smart' recognition capabilities, and which display functional peptide sequences. These smart and biofunctional streptavidin derivatives can 'talk' to cells, and 'listen' to external signals which control capture and release of biotinylated molecules.

Affinity Labels↗
PubMed1999

Development of new biotin/streptavidin reagents for pretargeting.

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.

Affinity Labels↗
PubMed1999

X-ray crystallographic studies of streptavidin mutants binding to biotin.

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.

Binding Sites↗
PubMed1999

Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics.

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.

Crystallization↗
PubMed1999

The design and synthesis of polymers for eukaryotic membrane disruption.

The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by one to two pH units, depending on the stage of endosomal development. This pH gradient is a key factor in the design of membrane-disruptive polymers which could enhance the endosomal release of drugs. Such polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We have designed and synthesized pH-sensitive synthetic polymers which efficiently disrupt red blood cells within a sharply defined pH range. One of these polymers, poly(ethyl acrylic acid) (PEAAc) has been previously shown to disrupt synthetic vesicles in a pH-dependent fashion [6]. PEAAc hemolyzes red blood cells with an activity of 10(7) molecules per red blood cell, which is as efficient on a molar basis as the peptide melittin. The mechanism of RBC hemolysis by PEAAc is consistent with the colloid osmotic mechanism. PEAAc's hemolytic activity rises rapidly as the pH decreases from 6.3 to 5.0, and there is no hemolytic activity at pH 7.4. A related polymer, poly(propyl acrylic acid) (PPAAc), was synthesized to test whether making the pendant alkyl group more hydrophobic by adding one methylene group would increase the hemolytic activity. PPAAc was found to disrupt red blood cells 15 times more efficiently than PEAAc at pH 6.1. PPAAc was also not active at pH 7.4 and displayed a pH-dependent hemolysis that was shifted toward higher pH's. Random 1:1 copolymers of ethyl acrylate (EA) and acrylic acid (AAc) (which contain random -COOH and -C(2)H(5) groups that are present and regularly repeat in PEAAc) also displayed significant hemolytic activity, with an efficiency close to PEAAc. These results demonstrate that pH-sensitive synthetic polymers can be molecularly engineered to efficiently disrupt eukaryotic membranes within defined and narrow pH ranges. Thus, these polymers might serve as endosomal disruptive agents with specificities for early or late endosomes.

Cell Membrane↗
PubMed1999

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Biotin↗
PubMed1999

Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs.

Saliva is a supersaturated solution with respect to hydroxyapatite, the main inorganic component of tooth enamel. Several acidic phosphoproteins are present in saliva which allow the supersaturated state to be maintained without random crystallization occurring. Statherin is the only salivary protein currently known to inhibit both the primary and secondary precipitation of hydroxyapatite in the supersaturated environment of saliva. To identify the residues of statherin that are necessary to control biomineralization, a recombinant form of human statherin was produced from Escherichia coli using a yeast intein fusion construct. The primary structure of the recombinant statherin was characterized by SDS-PAGE, N-terminus sequencing, MALDI mass spectrometry, and amino acid analysis and found to have the expected values relative to human-derived statherin. The secondary structure of the recombinant statherin was investigated by circular dichroism spectroscopy, which revealed the predominant presence of random coil in phosphate-buffered saline solution, with a higher propensity toward alpha helicity in 100% TFE. This increase in helicity in 100% TFE was also found in statherin that was synthesized by solid-phase synthesis. These results demonstrate that human statherin can be produced in a recombinant form which behaves comparably to the natural form.

Amino Acid Sequence↗