Linda A. Luck

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.

X-Ray Structures of the Leucine-Binding Protein Illustrate Conformational Changes and the Basis of Ligand Specificity

The periplasmic leucine-binding protein is the primary receptor for the leucine transport system in Escherichia coli. We report here the structure of an open ligand-free form solved by molecular replacement and refined at 1.5-Å resolution. In addition, two closed ligand-bound structures of the same protein are presented, a phenylalanine-bound form at 1.8 Å and a leucine-bound structure at a nominal resolution of 2.4 Å. These structures show the basis of this proteins ligand specificity, as well as illustrating the conformational changes that are associated with ligand binding. Comparison with earlier structures provides further information about solution conformations, as well as the different specificity of the closely related leucine/isoleucine/valine-binding protein.

Electrochemical Impedance Biosensor for Glucose Detection Utilizing a Periplasmic E. coli Receptor Protein

We report the development of a reagentless electrochemical impedance biosensor for glucose that employs the D-glucose/galactose receptor from E. coli for direct glucose detection. The biological platform for this sensor is an Au surface to which the protein is immobilized through formation of an Au-S bond to a genetically engineered cysteine residue at the N-terminus. The impedance signal detects the extensive ligand-induced domain motion within the protein upon glucose binding. We show the applicability of impedance spectroscopy in conjunction with periplasmic binding proteins as a general method for detecting small molecules.

19F NMR Studies of the Leucine-Isoleucine-Valine Binding Protein: Evidence That a Closed Conformation Exists in Solution

Abstract The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This “venus fly trap” type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential 19F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The 19F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.

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.

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.

Immobilization of the Glucose-Galactose Receptor Protein onto a Au Electrode Through a Genetically Engineered Cysteine Residue

Studies by electrochemical impedance spectroscopy demonstrate that the wild-type glucose-galactose receptor (GGR) protein does not interact significantly with a Au surface, even through nonspecific interactions. Only the GGR A1C mutant, where alanine is replaced by a cysteine residue at the N-terminus, can form a protein film on Au suitable for creating an impedance biosensor. Impedance measurements of glucose binding at equilibrium allow estimation of the dissociation constant (K d ) as approximately 6.6 (1.5) μM, about 32 times higher than that for the native protein in solution. This change likely arises from surface immobilization rather than from cysteine introduction.

The Wittig reaction in the generation of organometallic compounds containing alkenes as side groups

Abstract The Wittig reaction has been identified as a viable route to transition metal monomers. It has been used to synthesize {η 5 -C 5 [C(CH 3 )CHR]}Mn(CO) 3 [R  H (68% yield), -CH 3 (60%), -CH 2 CH 3 (51%), -CH 2 CH 2 CH 3 (40%), -C 6 H 5 (46%)] from acetylcymantrene and the appropriate phosphorane at room temperature. {η 5 C 5 H 4 [C(CH 3 )CHR]}(η 5 -C 5 H 5 )Fe [R  -H (81%), -CH 3 (77%). -CH 2 CH 3 (36%), -CH 2 CH 2 CH 3 (27%) have been prepared from acetylferrocene and phosphorane at room temperature. [η 5 -C 5 (CHCRR′)H 4 ](η 5 -C 5 -H 5 )Fe [R,R′ -H,H (79%); -CH 3 ,H (69%); -CH 2 CH 3 ,H (48%); -CH 2 CH 2 CH 3 ,H (49%); -C 6 H 5 ,H (80%); -C(CH 3 ) 2 H,H (73%); -CH 3 ,CH 3 (67%)] have been produced from formylferrocene and phosphorane in refluxing benzene. E / Z isomeric ratios were identified for alkenylcymantrenes and are consistent with past Wittig studies. The aldol reaction has been identified as a side route in the Wittig reactions of acetylferrocene and phosphoranes. Carbomethoxyphosphoranes did not produce alkenes at room temperature with nonpolar solvents.

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor.

19F nuclear magnetic resonance (19F NMR) of 5‐fluorotryptophan (5F‐Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F‐Trp. 19F NMR assignments for the 5F‐Trp residues (14, 25, 45, and 158) were based on comparison of the wild‐type protein spectrum with the spectra of three single Trp‐to‐Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild‐type sTF that the side chains of Trp14 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J1995;69:20–29). 19F NMR paramagnetic broadening and solvent‐induced isotope‐shift experiments show that position 5 of the indole ring of 5F‐Trp158 is exposed, whereas that of 5F‐Trp45 is essentially inaccessible. Although 5F‐Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild‐type or mutant proteins, 19F NMR chemical shifts showed that the single‐Trp mutations are accompanied by subtle changes in the local environments of 5F‐Trp residues residing in the same structural domain. Proteins 1999;37:709–716. ©1999 Wiley‐Liss, Inc.

NMR methods for analysis of CRALBP retinoid binding

Publisher Summary The identification of the cellular retinaldehyde-binding protein (CRALBP) retinoid-binding pocket and definition of the structural properties of the protein that provide high ligand stereoselectivity and low photosensitivity has been a prime matter of interest. In this purpose, solution state nuclear magnetic resonance (NMR) analysis has been initiated using human recombinant CRALBP labeled by biosynthetic isotope incorporation. A combination of heteronuclear gradient-enhanced 15N NMR and one-dimensional 19F and 13C NMR methods coupled with improved isotope incorporation methods and mass spectrometry, has proven to be complimentary approaches for characterizing CRALBP-ligand interactions. While these methods have been used separately elsewhere, usually as a primary approach to structural problems, this chapter emphasizes the complimentarity of the techniques and the advantages of combining the methods for studying protein–ligand interactions. Such protein biotechnology is suitable for characterizing a variety of protein–igand interactions and is becoming more accessible through specialized biomolecular resource facilities. This study describes the applicability of 15N, 19F, and 13C NMR methodology for studying ligand interactions in a light sensitive protein, such as recombinant CRALBP (rCRALBP). Gradient enhanced sensitivity enhanced heteronuclear single quantum correlation 15N NMR has provided evidence that rCRALBP undergoes a specific localized conformational change upon photoisomerization of 11-cis-retinaldehyde and removal of the ligand from the binding pocket. The results from the multidimensional NMR measurements strongly support the likelihood that the 19F Trp and 13C-Met NMR chemical shift differences observed for the protein with and without bound 11-cis-retinaldehyde are associated with protein–ligand interactions. Site directed mutagenesis in conjunction with further NMR and ligand binding studies assures identification of components of the rCRALBP retinoid binding pocket.