Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK's, Progressions, Quenching and Exciton Interaction.

A novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel 'q-deformed' type at various values of pH, and protein concentration.

Chasing Mendel: five questions for personalized medicine.

Ideas about personalized medicine are underpinned in part by evolutionary biology's Modern Synthesis. In this essay we link personalized medicine to the efforts of the early statistical investigators who quantified the heritability of human phenotype and then attempted to reconcile their observations with Mendelian genetics. As information about the heritability of common diseases was obtained, similar efforts were directed at understanding the genetic basis of disease phenotypes.

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds.

The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals.

Detailed structural-functional analysis of the Krüppel-like factor 16 (KLF16) transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology.

Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes).

Validation of fractal-like kinetic models by time-resolved binding kinetics of dansylamide and carbonic anhydrase in crowded media.

Kinetic studies of biochemical reactions are typically carried out in a dilute solution that rarely contains anything more than reactants, products, and buffers. In such studies, mass-action-based kinetic models are used to analyze the progress curves. However, intracellular compartments are crowded by macromolecules.

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins.

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold.

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy.

Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated.

Calmodulin wraps around its binding domain in the plasma membrane Ca2+ pump anchored by a novel 18-1 motif.

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.

Directly observed hydrogen bonds at calcium-binding-sites of calmodulin in solution relate to affinity of the calcium-binding.

Molecular tuning to calcium-binding in the EF-hand motif of holo-calmodulin was studied in solution by NMR (h3)J(NC') H-bond couplings. In the N-terminus lobe of holo-calmodulin, the glutamate crucial for Ca(2+) coordination has network of H-bonds weaker than inferred from the X-ray crystal structure. This glutamate at position 12 appears shifted away from the Ca(2+) preferred coordination, which can explain the lower affinity of the calcium-binding to the N-terminus with respect to C-terminus EF hands.

Dendritic cell microvilli: a novel membrane structure associated with the multifocal synapse and T-cell clustering.

Polarizing effects of productive dendritic cell (DC)-T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli.

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands.

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients.

Mathematical analysis of models for reaction kinetics in intracellular environments.

Two models that have been proposed in the literature for description of kinetics in intracellular environments characterized by macromolecular crowding and inhomogeneities, are mathematically analyzed and discussed. The models are first derived by using phenomenological arguments that lead to generalizations of the law of mass action. The prediction of these models in the case of bimolecular binding reaction is then analyzed.

Immunohistochemical expression of folate receptor alpha in colorectal carcinoma: patterns and biological significance.

Folate receptor alpha (FRalpha) has emerged as a potential cancer therapy target with several folate-linked therapeutic agents currently undergoing clinical trials. In addition, FRalpha expression in tumors may offer prognostic significance. Most studies on FRalpha expression used reverse transcriptase polymerase chain reaction or cytofluorimetric assays.

Structural dependencies of protein backbone 2JNC' couplings.

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide.

Solvent-induced differentiation of protein backbone hydrogen bonds in calmodulin.

In apo and holoCaM, almost half of the hydrogen bonds (H-bonds) at the protein backbone expected from the corresponding NMR or X-ray structures were not detected by h3JNC' couplings. The paucity of the h3JNC' couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein-backbone H-bonds form two groups according to the h3JNC' couplings measured in solution.

Photophysics of ANS. I. Protein-ANS complexes: Intestinal fatty acid binding protein and single-trp mutants.

We continue investigations into the physical chemistry of intestinal fatty acid binding protein, I-FABP, and its interaction with ANS and other ligands [cf references [Kirk, W., E. Kurian, and F.

Calcium-binding proteins afford calibration of dihedral-angle dependence of 3J(NC(gamma)) coupling constant in aspartate and asparagine residues.

Calibration of the 3J(NC(gamma)) couplings across the N-C(alpha)-C(beta)-C(gamma) fragment of aspartate and asparagine residues is afforded by two interactions that produce fixed conformations of the side chains in solution. One is the binding of these side chains to calcium ions; the other is the H-bond interaction of these side chains with a backbone amide.

Measurement of intermolecular distances for the natural agonist Peptide docked at the cholecystokinin receptor expressed in situ using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer is a powerful biophysical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed.

Water and urea interactions with the native and unfolded forms of a beta-barrel protein.

A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use (17)O and (2)H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein.

H-bonding mediates polarization of peptide groups in folded proteins.

The carbon-nitrogen J-couplings in the hydrogen bonding chains of proteins show that H-bonding mediates peptide-group polarization, which results in the general reduction of peptide-group polarity of folded proteins in solution. The net effect is to make large regions of protein secondary structure, especially beta-sheets, intrinsically more hydrophobic, contributing thereby to overall stability of the tertiary structure.

Accuracy of protein secondary structure determination from circular dichroism spectra based on immunoglobulin examples.

Strong contribution of the aromatic amino acid side chain chromophores to the far-UV circular dichroism (CD) spectra substantially distorts a relatively weak CD signal originating from beta sheet, the main type of immunoglobulin secondary structure. In this study we compared the secondary structure calculated from the far-UV CD spectra with the X-ray data for three antibody Fab fragments. Calculations were performed with three different algorithms, using two sets of reference proteins.

Structural dependencies of h3JNC' scalar coupling in protein H-bond chains.

The H-bond ((h3)J(NC')) and peptide bond ((1)J(NC')) scalar couplings establish connectivity of the electronic structure in the H-bond chains of proteins. The correlated changes of (h3)J(NC') and (1)J(NC') couplings extend over several peptide groups in the chains. Consequently, the electronic structure of the H-bond chains can affect (h3)J(NC') in a manner that is independent of the local H-bond geometry.

EUDOC: a computer program for identification of drug interaction sites in macromolecules and drug leads from chemical databases.

The completion of the Human Genome Project, the growing effort on proteomics, and the Structural Genomics Initiative have recently intensified the attention being paid to reliable computer docking programs able to identify molecules that can affect the function of a macromolecule through molecular complexation. We report herein an automated computer docking program, EUDOC, for prediction of ligand-receptor complexes from 3D receptor structures, including metalloproteins, and for identification of a subset enriched in drug leads from chemical databases. This program was evaluated from the standpoints of force field and sampling issues using 154 experimentally determined ligand-receptor complexes and four "real-life" applications of the EUDOC program.