Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: a glucose biosensor application.

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.

Nanobiosensor design utilizing a periplasmic E. coli receptor protein immobilized within Au/polycarbonate nanopores.

A new type of nanopore sensor design is reported for a reagent-less electrochemical biosensor with no analyte "tagging" by fluorescent molecules, nanoparticles, or other species. This sensor design involves immobilization within Au-coated nanopores of bacterial periplasmic binding proteins (bPBP), which undergo a wide-amplitude, hinge-twist motion upon ligand binding. Ligand binding thus triggers a reduction in the effective thickness of the immobilized protein film, which is detected as an increase in electrolyte conductivity (decrease in impedance) through the nanopores. This new sensor design is demonstrated for glucose detection using a cysteine-tagged mutant (GGR Q26C) of the galactose/glucose receptor (GGR) protein from the bPBP family. The GGR Q26C protein is immobilized onto Au nanoislands that are deposited within the pores of commercially available nanoporous polycarbonate membranes.

Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.

Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.

Molecular modeling of estrogen receptor using molecular operating environment.

Molecular modeling is pervasive in the pharmaceutical industry that employs many of our students from Biology, Chemistry and the interdisciplinary majors. To expose our students to this important aspect of their education we have incorporated a set of tutorials in our Biochemistry class. The present article describes one of our tutorials where undergraduates use modeling experiments to explore the structure of an estrogen receptor. We have employed the Molecular Operating Environment, a powerful molecular visualization software, which can be implemented on a variety of operating platforms. This tutorial reinforces the concepts of ligand binding, hydrophobicity, hydrogen bonding, and the properties of side chains and secondary structure taught in a general biochemistry class utilizing a protein that has importance in human biology.

A biosensor for estrogenic substances using the quartz crystal microbalance.

This article describes a biosensor that detects estrogenic substances using a quartz crystal microbalance with a genetically engineered construct of the hormone-binding domain of the alpha-estrogen receptor. The receptor was immobilized to a piezoelectric quartz crystal via a single exposed cysteine, forming a uniform orientation on the crystal surface. Our results illustrate that this sensor responds to a variety of ligands that are known to bind to the estrogen receptor. No response was observed for nonbinding substances such as testosterone and progesterone. The sensitive response of this biosensor to estrogenic substances results from changes in the structural rigidity of the immobilized receptor that occurs with ligand binding. Agonist and antagonist show different responses.

A piezoelectric quartz crystal biosensor: the use of two single cysteine mutants of the periplasmic Escherichia coli glucose/galactose receptor as target proteins for the detection of glucose.

We have examined the potential utility of a glucose biosensor that employs the glucose/galactose receptor of Escherichia coli with a quartz crystal microbalance (QCM). Two different genetically engineered mutant proteins were utilized, each involving the incorporation of a single cysteine into the amino acid sequence of the protein. The proteins were immobilized on the surface of a piezoelectric crystal by a direct sulfur-gold linkage. Since the cysteines were located at different positions in the sequence, the receptors attach to the surface with different orientations. Considering only mass effects, the target sugars for this receptor are predicted to be too small to be detectable with a QCM. However, our sensors indicated measurable and reproducible frequency responses when immobilized receptor was exposed to sugar. This unexpectedly large frequency response occurs because the protein film is transformed from a viscous layer to a more rigid nondissipative film. The QCM can detect these changes because of the direct linkage of the proteins to the surface. Calculations of the frequency response expected for a viscoelastic film with different rheological characteristics support this hypothesis. This study is significant because it illustrates a widened applicability for the QCM methodology to protein systems that bind small molecules and undergo ligand-induced conformational changes.

Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins.

This paper demonstrates potential applications of two periplasmic receptor proteins from E. coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique. These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers. Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics. We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands. According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model. The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work.

Quantitative analysis of tryptophan analogue incorporation in recombinant proteins.

Three different methods to quantitate tryptophan (Trp) analogue incorporation into recombinant proteins are described: first, spectroscopic analysis based on a linear combination of the absorption spectra of the aromatic residues in the denatured Trp-containing or analogue-substituted protein; second, chromatographic separation of analogue-substituted and Trp-containing proteins by HPLC; and third, mass spectrum analysis of the mixture of analogue-substituted and Trp-containing proteins. An accurate estimate of analogue incorporation in single-Trp proteins can be obtained directly by either analysis of the absorption spectrum or HPLC chromatography. While analysis of the absorption spectrum or HPLC chromatogram can provide an assessment of the average level of analogue incorporation for proteins that contain two or more Trp residues, mass spectroscopy analysis of peptides generated by protease digestion and separated by HPLC provides a general method for a complete quantitative description of the distribution of analogue incorporation. The more complex analysis by mass spectroscopy becomes important for multi-Trp proteins because the distribution of analogue versus Trp-containing polypeptide chains may not be the same as that predicted on the basis of average level of analogue incorporation.