Characterization of the mechanism of interaction between α -acid glycoprotein and lipid membranes by vacuum-ultraviolet circular-dichroism spectroscopy.

α -Acid glycoprotein (AGP) interacts with lipid membranes as a peripheral membrane protein so as to decrease the drug-binding capacity accompanying the β→α conformational change that is considered a protein-mediated uptake mechanism for releasing drugs into membranes or cells. This study characterized the mechanism of interaction between AGP and lipid membranes by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of AGP down to 170 nm using synchrotron radiation in the presence of five types of liposomes whose constituent phospholipid molecules have different molecular characteristics in the head groups (e.g.

Vacuum Ultraviolet Electronic Circular Dichroism Study of d-Glucose in Aqueous Solution.

Vacuum ultraviolet (VUV) electronic circular dichroism (ECD) spectra of d-glucose, α-d-glucopyranose, and β-d-glucopyranose were measured in aqueous solution down to 163 nm using a synchrotron radiation VUV-ECD spectrophotometer and theoretically analyzed using molecular dynamics (MD) simulations with explicit water molecules and using time-dependent density functional theory (TDDFT). The theoretically calculated spectra reproduced the experimentally observed spectra well, revealing that VUV-ECD exhibited unique spectra depending on the α-anomer and β-anomer configurations of the hydroxyl group at C-1 and the three gauche (G) and trans (T) rotamer conformations (GT, GG, and TG) of the hydroxymethyl group at C-5. These unique spectra could be ascribed to differences in the patterns of intramolecular hydrogen bonds around the hydroxymethyl group at C-5 for the three rotamers and around the hydroxyl group at C-1 for the two anomers.

Regulation of plant ER oxidoreductin 1 (ERO1) activity for efficient oxidative protein folding.

In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1-PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins.

Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Lipid Environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190 nm and producing information that is unobtainable by conventional CD instruments.

Synchrotron-radiation vacuum-ultraviolet circular dichroism spectroscopy in structural biology: an overview.

Circular dichroism spectroscopy is widely used for analyzing the structures of chiral molecules, including biomolecules. Vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy using synchrotron radiation can extend the short-wavelength limit into the vacuum-ultraviolet region (down to ~160 nm) to provide detailed and new information about the structures of biomolecules in combination with theoretical analysis and bioinformatics. The VUVCD spectra of saccharides can detect the high-energy transitions of chromophores such as hydroxy and acetal groups, disclosing the contributions of inter- or intramolecular hydrogen bonds to the equilibrium configuration of monosaccharides in aqueous solution.

Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectroscopy for Characterizing the Structure of Saccharides.

Circular-dichroism (CD) spectroscopy is a powerful tool for analyzing the structures of chiral molecules and biomolecules. The development of CD instruments using synchrotron radiation has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet (VUV) region below 190 nm and thereby yielding information that is unobtainable by conventional CD instruments. This technique is especially advantageous for monitoring the structure of saccharides that contain hydroxy and acetal groups with high-energy transitions in the VUV region.

Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy.

A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement.

Conformation of membrane-bound proteins revealed by vacuum-ultraviolet circular-dichroism and linear-dichroism spectroscopy.

Knowledge of the conformations of a water-soluble protein bound to a membrane is important for understanding the membrane-interaction mechanisms and the membrane-mediated functions of the protein. In this study we applied vacuum-ultraviolet circular-dichroism (VUVCD) and linear-dichroism (LD) spectroscopy to analyze the conformations of α-lactalbumin (LA), thioredoxin (Trx), and β-lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural-network analysis showed that these three proteins have characteristic helix-rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome.

Isotope effect on the circular dichroism spectrum of methyl α-D-glucopyranoside in aqueous solution.

H/D isotope effect on the circular dichroism spectrum of methyl α-D-glucopyranoside in aqueous solution has been analyzed by multicomponent density functional theory calculations using the polarizable continuum model. By comparing the computational spectra with the corresponding experimental spectrum obtained with a vacuum-ultraviolet circular dichroism spectrophotometer, it was demonstrated that the isotope effect provides insights not only into the isotopic difference of the intramolecular interactions of the solutes, but also into that of the solute-solvent intermolecular interaction.

Environmental Adaptation of Dihydrofolate Reductase from Deep-Sea Bacteria.

In order to elucidate the molecular adaptation mechanisms of enzymes to the high hydrostatic pressure of the deep sea, we cloned, purified, and characterized more than ten dihydrofolate reductases (DHFRs) from bacteria living in deep-sea and ambient atmospheric pressure environments. The nucleotide and amino acid sequences of these DHFRs indicate the deep-sea bacteria are adapted to their environments after the differentiation of their genus from ancestors inhabiting atmospheric pressure environments. In particular, the backbone structure of the deep-sea DHFR from Moritella profunda (mpDHFR) almost overlapped with the normal homolog from Escherichia coli (ecDHFR).

Volume and Compressibility of Proteins.

The partial specific (or molar) volume, expansibility, and compressibility of a protein are fundamental thermodynamic quantities for characterizing its structure in solution. We review the definitions, measurements, and implications of these volumetric quantities in relation to protein structural biology. The partial specific volumes under constant molality (isomolal) and chemical potential (isopotential) conditions of the cosolvent (multicomponent systems) are explained in terms of preferential solvent interactions relevant to the solubility and stability of proteins.

Characterization of intermolecular structure of β(2)-microglobulin core fragments in amyloid fibrils by vacuum-ultraviolet circular dichroism spectroscopy and circular dichroism theory.

Intermolecular structures are important factors for understanding the conformational properties of amyloid fibrils. In this study, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy and circular dichroism (CD) theory were used for characterizing the intermolecular structures of β2-microglobulin (β2m) core fragments in the amyloid fibrils. The VUVCD spectra of β2m20-41, β2m21-31, and β2m21-29 fragments in the amyloid fibrils exhibited characteristic features, but they were affected not only by the backbone conformations but also by the aromatic side-chain conformations.

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop.

Circular-dichroism and synchrotron-radiation circular-dichroism spectroscopy as tools to monitor protein structure in a lipid environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190 nm and producing information that is unobtainable by conventional CD instruments.

Vacuum-ultraviolet electronic circular dichroism study of methyl α-D-glucopyranoside in aqueous solution by time-dependent density functional theory.

The vacuum-ultraviolet (VUV) electronic circular dichroism (ECD) spectrum of methyl α-D-glucopyranoside (methyl α-D-Glc) was measured down to 163 nm in aqueous solution using a synchrotron-radiation VUV-ECD spectrophotometer. The spectrum exhibited two characteristic ECD peaks around 170 nm, which depend on the trans (T) and gauche (G) configurations of the hydroxymethyl group at C-5. To elucidate the influences of the T and G configurations on the spectrum, the ECD spectra of three rotamers (α-GT, α-GG, and α-TG) of methyl α-D-Glc were calculated using time-dependent density functional theory (TDDFT) combined with molecular dynamics simulation.

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli.

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra.

Nonneutral evolution of volume fluctuations in lysozymes revealed by normal-mode analysis of compressibility.

The evolution of structural fluctuations of proteins was examined by calculating the isothermal compressibility (β(T)) values of chicken lysozyme and its six evolutionary mutants at Thr40, Ile55, and Ser91 (a ternary mutant corresponding to bobwhite lysozyme) from their X-ray structures by normal-mode analysis at 300 K. The β(T) values of the two extant lysozymes from chicken and bobwhite were 1.61 and 1.

Secondary-structure analysis of alcohol-denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy.

To elucidate the structural characteristics of alcohol-denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six proteins-myoglobin, human serum albumin, α-lactalbumin, thioredoxin, β-lactoglobulin, and α-chymotrypsinogen A-down to 170 nm in trifluoroethanol solutions (TFE: 0-50%) and down to 175 nm in methanol solutions (MeOH: 0-70%) at pH 2.0 and 25°C, using a synchrotron-radiation VUVCD spectrophotometer. The contents of α-helices, β-strands, turns, poly-L-proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α-helix and β-strand segments.

Experimental and theoretical studies of vacuum-ultraviolet electronic circular dichroism of hydroxy acids in aqueous solution.

The electronic circular dichroism (ECD) spectra of three L-hydroxy acids (L-lactic acid, (+)-(S)-2-hydroxy-3-methylbutyric acid, and (-)-(S)-2-hydroxyisocaproic acid) were measured down to 160 nm in aqueous solution using a vacuum-ultraviolet ECD spectrophotometer. To assign the two positive peaks around 210 and 175 nm and the one negative peak around 190 nm in the observed spectra, the ECD spectrum of L-lactic acid was calculated using time-dependent density functional theory (DFT) for the optimized structures by DFT and a continuum model. The observed ECD spectrum was successfully reproduced as the average spectrum for four optimized structures with seven water molecules that localized around the COO(-) and OH groups of L-lactic acid.

Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles.

Clusters of GM1 gangliosides act as platforms for conformational transition of monomeric, unstructured amyloid β (Aβ) to its toxic β-structured aggregates. We have previously shown that Aβ(1-40) accommodated on the hydrophobic/hydrophilic interface of lyso-GM1 or GM1 micelles assumes α-helical structures under ganglioside-excess conditions. For better understanding of the mechanisms underlying the α-to-β conformational transition of Aβ on GM1 clusters, we performed spectroscopic characterization of Aβ(1-40) titrated with GM1.

Comparative study on dihydrofolate reductases from Shewanella species living in deep-sea and ambient atmospheric-pressure environments.

To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74-90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR).

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants.

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants.

Cloning and characterization of dihydrofolate reductases from deep-sea bacteria.

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different.

Differences in the molecular structure of beta(2)-microglobulin between two morphologically different amyloid fibrils.

Differences in the molecular structures of beta(2)-microglobulin between the two morphologically different amyloid fibrils having a needlelike [long-straight (LS)] and flexible [wormlike (WL)] character were investigated by infrared, Raman, and vacuum-ultraviolet circular dichroism spectroscopy. It turned out that although the beta-sheet content was comparable between the two kinds of fibrils (53 +/- 6% for the LS fibril and 47 +/- 6% for the WL fibril), the protonation states of the carboxyl side chains were distinctly different; the deprotonated (COO(-)) and protonated (COOH) forms were dominant in the LS and WL fibrils at pH 2.5, respectively, meaning that the pK(a) is specifically lowered in the LS fibril.