Characterization of the mechanism of interaction between α1 -acid glycoprotein and lipid membranes by vacuum-ultraviolet circular-dichroism spectroscopy.

α1 -Acid glycoprotein (AGP) interacts with lipid membranes as a peripheral membrane protein so as to decrease the drug-binding capacity accompanying the ß→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., different net charges). The VUVCD analysis showed that the α-helix and ß-strand contents and the numbers of segments of AGP varied with the constituent phospholipid molecules of liposomes, while combining VUVCD data with a neural-network method predicted that these membrane-bound conformations comprised several common long helix and small strand segments. The amino-acid composition of each helical segment of the conformations indicated that amphiphilic and positively charged helices formed at the N- and C-terminal regions of AGP, respectively, were candidate sites for the membrane interaction. The addition of 1 M sodium chloride shortened the C-terminal helix while having no effect on the length of the N-terminal one. These results suggest that the N- and C-terminal helices can interact with the membrane via hydrophobic and electrostatic interactions, respectively, demonstrating that the liposome-dependent conformations of AGP analyzed using VUVCD spectroscopy provide useful information for characterizing the mechanism of interaction between AGP and lipid membranes.

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 ß-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 ß-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. The strengths of these intramolecular interactions increased as the degree of hydration around the corresponding chromophores decreased, suggesting that hydration is a key factor for stabilizing rotamer and anomer structures. The rotamerization and anomerization mechanisms are further discussed in terms of differences in the intramolecular interactions and the degree of hydration among the rotamer and anomer structures. The findings demonstrate that VUV-ECD spectroscopy is a useful tool for characterizing the equilibrium structures of monosaccharides.

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. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II 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. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

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. The roles of hydration in the fluctuation of the dihedral angles of carboxyl and amino groups of amino acids can be clarified by comparing the observed VUVCD spectra with those calculated theoretically. The VUVCD spectra of proteins markedly improves the accuracy of predicting the contents and number of segments of the secondary structures, and their amino acid sequences when combined with bioinformatics, for not only native but also nonnative and membrane-bound proteins. The VUVCD spectra of nucleic acids confirm the contributions of the base composition and sequence to the conformation in comparative analyses of synthetic poly-nucleotides composed of selected bases. This review surveys these recent applications of synchrotron-radiation VUVCD spectroscopy in structural biology, covering saccharides, amino acids, proteins, and nucleic acids.

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. Combining VUVCD spectra with theoretical calculations provides new insight into the contributions of anomeric hydroxy groups and rotational isomers of hydroxymethyl groups to the dynamics, intramolecular hydrogen bonds, and hydration of saccharides in aqueous solution.

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. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies.

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 ß-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. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence-level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane-binding proteins, which are difficult targets for X-ray crystallography and nuclear magnetic resonance spectroscopy.

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.

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. The partial expansibility is briefly discussed in terms of the effects of temperature on protein-solvent interactions (hydration) and internal packing defects (cavities). We discuss the compressibility-structure-function relationships of proteins based on analyses of the correlations between the partial adiabatic compressibilities and the structures or functions of various globular proteins (including mutants), focusing on the roles of the internal cavities in structural fluctuations. The volume and compressibility changes associated with various conformational transitions are also discussed in terms of the changes in hydration and cavities in order to elucidate the nonnative structures and the transition mechanisms, especially those associated with pressure denaturation.

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). Thus, those of other DHFRs would also overlap on the basis of their sequence similarities. However, the structural stability of both DHFRs was quite different: compared to ecDHFR, mpDHFR was more thermally stable but less stable against urea and pressure unfolding. The smaller volume changes due to unfolding suggest that the native structure of mpDHFR has a smaller cavity and/or enhanced hydration compared to ecDHFR. High hydrostatic pressure reduced the enzymatic activity of many DHFRs, but three deep-sea DHFRs and the D27E mutant of ecDHFR exhibited pressure-dependent activation. The inverted activation volumes from positive to negative values indicate the modification of their structural dynamics, conversion of the rate-determining step of the enzymatic reaction, and different contributions of the cavity and hydration to the transition-state structure. Since the cavity and hydration depend on amino acid side chains, DHFRs would adapt to the deep-sea environment by regulating the cavity and hydration by substituting their amino acid side chains without altering their backbone structure. The results of this study clearly indicate that the cavity and hydration play important roles in the adaptation of enzymes to the deep-sea environment.

Characterization of intermolecular structure of ß(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 ß2-microglobulin (ß2m) core fragments in the amyloid fibrils. The VUVCD spectra of ß2m20-41, ß2m21-31, and ß2m21-29 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. To estimate the contributions of aromatic side-chains to the spectra, the theoretical spectra were calculated from the simulated structures of ß2m21-29 amyloid fibrils with various types of ß-sheet stacking (parallel or antiparallel) using CD theory. We found that the experimental spectrum of ß2m21-29 fibrils is largely affected by aromatic-backbone couplings, which are induced by the interaction between transitions within the aromatic and backbone chromophores, and these couplings are sensitive to the type of stacking among the ß-sheets of the fibrils. Further theoretical analyses of simulated structures incorporating mutated aromatic residues suggested that the ß2m21-29 fibrils are composed of amyloid accumulations in which the parallel ß-sheets stack in an antiparallel manner and that the characteristic Phe-Tyr interactions among the ß-sheet stacks affect the aromatic-backbone coupling. These findings indicate that the coupling components, which depend on the characteristic intermolecular structures, induce the spectral differences among three fragments in the amyloid fibrils. These advanced spectral analyses using CD theory provide a useful method for characterizing the intermolecular structures of protein and peptide fragment complexes.

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. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.

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. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

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. A linear combination of the ECD spectra of these three rotamers, which differ markedly from each other, produced a methyl α-D-Glc spectrum similar to that observed experimentally. The spectrum was assignable to the n-σ* transitions of the ring oxygen and methoxy oxygen with minor contributions from the hydroxyl oxygen. The differences in α-GT, α-GG, and α-TG spectra were attributed to fluctuations of the configurations of the hydroxymethyl group at C-5 and the hydroxyl group at C-4, which strongly affected the orientations of intramolecular hydrogen bonds around the ring oxygen. These findings demonstrate that combining VUV-ECD and TDDFT is useful for structural characterization of saccharides in aqueous solution.

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. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

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 (ß(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 ß(T) values of the two extant lysozymes from chicken and bobwhite were 1.61 and 1.59 Mbar(-1), respectively, but five other evolutionary mutants showed larger ß(T) values of up to 2.17 Mbar(-1). These results suggest that ancestral lysozymes exhibit larger volume fluctuations than extant ones, and hence that the molecular evolution of lysozymes has followed a nonneutral evolutionary pathway. The evolutionary mutants contained large amount of cavities, although no change was visible in the X-ray structures. There was a linear correlation between ß(T) and total cavity volume, predicting that the cavity volume or atomic packing is an important factor regulating volume fluctuations during the molecular evolution of this protein.

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, ß-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, ß-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 ß-strand segments. Furthermore, the positions of α-helices and ß-strands on amino acid sequences were predicted by combining these secondary-structure data with a neural-network method. All alcohol-denatured proteins showed higher α-helix contents (up to ~ 90%) compared with the native states, and they consisted of several long helical segments. The helix-forming ability was higher in TFE than in MeOH, whereas small amounts of ß-strands without sheets were formed in the MeOH solution. The produced α-helices were transformed dominantly from the ß-strands and unordered structures, and slightly from the turns. The content and mean length of α-helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol-denatured proteins constitute an ensemble of many long α-helices, a few ß-strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved.

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. The positive peak around 210 nm and the negative peak around 185 nm in the calculated spectrum were attributable to the nπ* transition of the carboxyl group, with the latter peak also being influenced by the ππ* transition of the carboxyl group; however, the positive peak around 165 nm involved unassignable higher energy transitions. The comparison of the calculated ECD spectra for L-lactic acid and L-alanine revealed that the network with loose hydrogen bonding around the COO(-) and OH groups is responsible for the flexible conformation of hydroxy acids and complicated side-chain dependence of ECD spectra relative to amino acids.