Mass spectrometry of hydrogen/deuterium exchange in 70S ribosomal proteins from E. coli

AbstractThe 70S ribosome from Escherichia coli is a supermacro complex (MW: 2.7MDa) comprising three RNA molecules and more than 50 proteins. We have for the first time successfully analyzed the flexibility of 70S ribosomal proteins in solution by detecting the hydrogen/deuterium exchange with mass spectrometry. Based on the deuterium incorporation map of the X-ray structure obtained at the time of each exchange, we demonstrate the structure–flexibility–function relationship of ribosome focusing on the deuterium incorporation of the proteins binding ligands (tRNA, mRNA, and elongation factor) and the relation with structural assembly processes

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 (beta(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 Or 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 beta(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 beta(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. (C) 2011 Elsevier B.V. All rights reserved

Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea

Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5-7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions

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 alpha-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of alpha-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

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 call (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa 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 -53 ml/mol at 25 degrees C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85 ml/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

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 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., different net charges). The VUVCD analysis showed that the α-helix and β-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.This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (numbers 19K06587 and 15K07028).[This article is part of the Special Issue: In honor and memory of Prof. Koji Nakanishi. See the first articles for this special issue previously published in Volumes 31:12, 32:3, and 32:4. More special articles will be found in this issue as well as in those to come.

Pressure dependence of the apparent specific volume of bovine serum albumin : Insight into the difference between isothermal and adiabatic compressibilities

There are some theoretical arguments related to interpreting the adiabatic compressibility (beta(s)) of a protein determined from the sound velocity and the difference between beta(s) and isothermal compressibility (beta(T)). To address these problems experimentally, we constructed a high-pressure oscillating densitometer and used it to measure the apparent specific volume of bovine serum albumin as a function of pressure (0.1-78 MPa) and temperature (5-35 degrees C). The beta(T) determined from plots of the apparent specific volume vs. pressure was slightly larger than beta(s) at all temperatures examined, with the difference between the two compressibilities increasing as the temperature was decreased. Only at room temperature did the observed beta(T) agree with those estimated from beta(s) using the heat capacity and the thermal expansibility of the protein, suggesting that there are significant as-yet-unknown mechanisms that affect protein compressibility