Check out our New Publishers' Select for Free Articles

BBA - Proteins and Proteomics (v.1804, #1)

Editorial Board (pp. i).

EuPa 2010 Young Investigator Award (pp. ii).

EuPa 2010 Senior investigator Award (pp. iii).

Reviewer Acknowledgement (pp. iv-vi).

EuPA 2010 Announcement (pp. i).

The Protein-Water Energy Seascape by Wolfgang Doster; Thomas Gutberlet (pp. 1-2).

The protein-solvent glass transition by Wolfgang Doster (pp. 3-14).

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G≈170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.

Keywords: Protein dynamics; Neutron scattering; Dynamical transition; Glass transition; Mode coupling theory; Protein hydration; Myoglobin; Lysozyme

A broad glass transition in hydrated proteins by S. Khodadadi; A. Malkovskiy; A. Kisliuk; A.P. Sokolov (pp. 15-19).

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g∼180±15 K. This result agrees with a broad range of T_g∼160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g∼180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement < r²> at temperatures around T_D∼200–230 K, and the glass transition. They have different physical origin and should not be confused.

Keywords: Hydrated protein; Glass transition temperature; Dynamic transition; Light scattering; Dielectric spectroscopy

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry by H. Jansson; J. Swenson (pp. 20-26).

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.

Keywords: Protein; Glass transition; Dielectric spectroscopy; Differential scanning calorimetry; Protein dynamics; Solvent dynamics

Effect of conformational states on protein dynamical transition by Hiroshi Nakagawa; Hironari Kamikubo; Mikio Kataoka (pp. 27-33).

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.

Keywords: Protein dynamic; Protein conformation; Inelastic neutron scattering; Hydration; Dynamical transition; Dynamical heterogeneity

Dynamical transition in a large globular protein: Macroscopic properties and glass transition by C.S. Kealley; A.V. Sokolova; G.J. Kearley; E. Kemner; M. Russina; A. Faraone; W.A. Hamilton; E.P. Gilbert (pp. 34-40).

Hydrated soy-proteins display different macroscopic properties below and above approximately 25% moisture. This is relevant to the food industry in terms of processing and handling. Quasi-elastic neutron spectroscopy of a large globular soy-protein, glycinin, reveals that a similar moisture-content dependence exists for the microscopic dynamics as well. We find evidence of a transition analogous to those found in smaller proteins, when investigated as a function of temperature, at the so-called dynamical transition. In contrast, the glass transition seems to be unrelated. Small proteins are good model systems for the much larger proteins because the relaxation characteristics are rather similar despite the change in scale. For dry samples, which do not show the dynamical transition, the dynamics of the methyl group is probably the most important contribution to the QENS spectra, however a simple rotational model is not able to explain the data. Our results indicate that the dynamics that occurs above the transition temperature is unrelated to that at lower temperatures and that the transition is not simply related to the relaxation rate falling within the spectral window of the spectrometer.

Keywords: Dynamic; Globular protein; Temperature; Moisture content

²H and¹³C NMR studies on the temperature-dependent water and protein dynamics in hydrated elastin, myoglobin and collagen by Sorin A. Lusceac; Michael R. Vogel ⁎; Claudia R. Herbers (pp. 41-48).

²H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h=0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K.¹³C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of²H and¹³C NMR data reveals that the observed protein dynamics is slower than the water dynamics.

Keywords: NMR; Water dynamics; Myoglobin; Elastin; Collagen

Motion characterization by self-distribution–function procedure by Salvatore Magazù; Giacomo Maisano; Federica Migliardo; Antonio Benedetto (pp. 49-55).

In the present paper a procedure for the biomolecular motion characterization based on the evaluation of the Mean Square Displacement (MSD), through the Self Distribution Function (SDF), is presented. In particular it will be shown how the MSD, which represents a good observable for the characterization of the dynamical properties in disordered systems, can be decomposed into partial contributions associated to the system dynamical processes within a specific spatial scale. It will be shown how the SDF procedure allows to evaluate both the total MSD and the partial MSDs through the total SFD and the partial SDFs. As a result, the total MSD is the weighed sum of the partial MSD contributions in which the weights are obtained by the fitting procedure of measured EINS intensity data. We apply the SDF procedure at EINS data collected, by the IN13 backscattering spectrometer at the Institute Laue-Langevin, Grenoble, on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry myoglobin in trehalose environment. It emerges that the hydrogen bond imposed network of the water–trehalose mixture appears to be stronger with respect to that of the water–sucrose mixture and this result can justify the highest bioprotectant effectiveness of trehalose in comparison with sucrose. Furthermore it emerges that, the partial MSD behaviours of sucrose and trehalose are equivalent in the low Q domain (0–1.7) Å−¹ whereas they are different in the high Q domain (1.7–4) Å^-¹. This circumstance suggests that the higher structure sensitivity of sucrose in respect to trehalose should be related to the small spatial observation windows.

Keywords: Molecular motion; Self-distribution–function; Mean square displacement; Resolution effect; Elastic incoherent neutron scattering; Homologous disaccharide

Self-similar dynamics of proteins under hydrostatic pressure—Computer simulations and experiments by G.R. Kneller; V. Calandrini (pp. 56-62).

Different experimental techniques, such as kinetic studies of ligand binding and fluorescence correlation spectroscopy, have revealed that the diffusive, internal dynamics of proteins exhibits autosimilarity on the time scale from microseconds to hours. Computer simulations have demonstrated that this type of dynamics is already established on the much shorter nanosecond time scale, which is also covered by quasielastic neutron scattering experiments. The autosimilarity of protein dynamics is reflected in long-time memory effects in the underlying diffusion processes, which lead to a non-exponential decay of the observed time correlation functions. Fractional Brownian dynamics is an empirical model which is able to capture the essential aspects of internal protein dynamics. Here we give a brief introduction into the theory and show how the model can be used to interpret neutron scattering experiments and molecular dynamics simulation of proteins in solution under hydrostatic pressure.

Keywords: Protein dynamics; Quasielastic neutron scattering; Hydrostatic pressure; Slow relaxation; Fractional Brownian dynamics

Elastic incoherent neutron scattering as a probe of high pressure induced changes in protein flexibility by A. Filabozzi; A. Deriu; M.T. Di Bari; D. Russo; S. Croci; A. Di Venere (pp. 63-67).

We report here the results of elastic incoherent neutron scattering experiments on three globular proteins (trypsin, lysozyme and β-lactoglobulin) in different pressure intervals ranging from 1 bar to 5.5 kbar. A decrease of the mean square hydrogen fluctuations, 〈 u²〉, has been observed upon increasing pressure. Trypsin and β-lactoglobulin behave similarly while lysozyme shows much larger changes in 〈 u²〉. This can be related to different steps in the denaturing processes and to the high propensity of lysozyme to form amyloids. Elastic incoherent neutron scattering has proven to be an effective microscopic technique for the investigation of pressure induced changes in protein flexibility.

Keywords: Neutron scattering; High pressure; Protein unfolding; Protein flexibility; Trypsin; Lysozyme; Beta-lactoglobulin

Protein diffusion in crowded electrolyte solutions by Felix Roosen-Runge; Marcus Hennig; Tilo Seydel ⁎; Fajun Zhang; Maximilian W.A. Skoda; Stefan Zorn; Robert M.J. Jacobs; Marco Maccarini; Peter Fouquet; Frank Schreiber (pp. 68-75).

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.

Keywords: Quasi-elastic neutron scattering; Cold neutron backscattering spectroscopy; Neutron spin echo spectroscopy; Globular proteins in aqueous solution; Salt ions

Using polarization analysis to separate the coherent and incoherent scattering from protein samples by Ana M. Gaspar ⁎; Sebastian Busch; Marie-Sousai Appavou; Wolfgang Haeussler; Robert Georgii; Yixi Su; Wolfgang Doster (pp. 76-82).

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006< Q<0.04 Å−¹) to the wide-angle region (up to ≈2.35 Å−¹). Quantitative information was obtained on the fraction of coherent to spin-incoherent scattering from different protein samples: deuterated and protonated protein powders at different hydration levels and solutions of protonated proteins in D₂O at different concentrations. The results obtained are discussed in the context of the validity of the assumptions generally made when interpreting quasielastic neutron scattering experiments performed without polarization analysis.

Keywords: Neutron scattering; Polarization analysis; Coherent/incoherent nuclear scattering; Protonated/deuterated protein; Structure; Dynamic

Time-resolved quasielastic neutron scattering studies of native photosystems by Jörg Pieper (pp. 83-88).

The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of ∼1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics ∼160 μs after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q_A→ Q_B electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.

Keywords: Time-resolved quasielastic neutron scattering; Protein dynamic; Bacteriorhodopsin; Photocycle; Photosynthesis; Photosystem II

The logic of the hepatic methionine metabolic cycle by M.V. Martinov; V.M. Vitvitsky; R. Banerjee; F.I. Ataullakhanov (pp. 89-96).

This review describes our current understanding of the “traffic lights” that regulate sulfur flow through the methionine bionetwork in liver, which supplies two major homeostatic systems governing cellular methylation and antioxidant potential. Theoretical concepts derived from mathematical modeling of this metabolic nexus provide insights into the properties of this system, some of which seem to be paradoxical at first glance. Cellular needs supported by this network are met by use of parallel metabolic tracks that are differentially controlled by intermediates in the pathway. A major task, i.e. providing cellular methylases with the methylating substrate, S-adenosylmethionine, is met by flux through the methionine adenosyltransferase I isoform. On the other hand, a second important function, i.e., stabilization of the blood methionine concentration in the face of high dietary intake of this amino acid, is achieved by switching to an alternative isoform, methionine adenosyltransferase III, and to glycine N-methyl transferase, which together bypass the first two reactions in the methionine cycle. This regulatory strategy leads to two metabolic modes that differ in metabolite concentrations and metabolic rates almost by an order of magnitude. Switching between these modes occurs in a narrow trigger zone of methionine concentration. Complementary experimental and theoretical analyses of hepatic methionine metabolism have been richly informative and have the potential to illuminate its response to oxidative challenge, to methionine restriction and lifespan extension studies and to diseases resulting from deficiencies at specific loci in this pathway.

Keywords: Abbreviations; AdoMet; S-adenosylmethionine; AdoHcy; S-adenosylhomocysteine; AHC; adenosylhomocysteinase; BHMT; betaine homocysteine S-methyltransferase; CBS; cystathionine β-synthase; GSH; glutathione; GNMT; glycine-N-methyltransferase; Hcy; homocysteine; MATI/II; III, methionine adenosyltransferase I/II,III; MGA; methyl group acceptor; MS; methionine synthase; MTHF; 5-methyltetrahydrofolate; MTHFR; methylenetetrahydrofolate reductaseMethionine metabolism; Sulfur, Mathematical model, Allostery

Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme by Russell A. Carpenter; Xuanzhi Zhan; Holly R. Ellis (pp. 97-105).

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH₂ binding. The k_cat/ K_m value for the C54S SsuD variant increased 3-fold, whereas the k_cat/ K_m value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH₂ was mixed against C54S SsuD ( k_obs, 111 s−¹) in oxygenated buffer that was 10-fold faster than wild-type SsuD ( k_obs, 12.9 s−¹). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.

Keywords: Abbreviations; FMN; flavin mononucleotide; FMNH; ₂; reduced flavin mononucleotide; NAD(P)H; nicotinamide adenine dinucleotide (phosphate); SsuE; alkanesulfonate flavin reductase; SsuD; alkanesulfonate monooxygenaseAlkanesulfonate monooxygenase; FMN reductase; SsuE; SsuD; Rapid reaction kinetic

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant by Panteha Mirarefi; C. Ted Lee Jr. (pp. 106-114).

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.

Keywords: Abbreviations; azoTAB; azobenzenetrimethlyammonium bromide; ANS; 1-anilino-8-naphthalenesulfonic acid; BSA; bovine serum albumin; CA; carbonic anhydrase; DTAB; dodecyltrimethylammonium bromide; DLS; dynamic light scattering; FSD; Fourier self-deconvolution, FT-IR, Fourier transform infrared; GuHCl; guanidine hydrochloride; MG; molten globule; PDDF; pair distance distribution function; SANS; small-angle neutron scattering; SAXS; small-angle X-ray scattering; SDS; sodium dodecyl sulfate; TFE; 2,2,2-trifluoroethanol; UV–vis; UV–visibleSmall-angle neutron scattering; Photoresponsive surfactant; Protein unfolding; Protein aggregation; Enzyme activity; Carbonic anhydrase

A new topology of ACBP from Moniliophthora perniciosa by Paulo S. Monzani; Humberto M. Pereira; Fernando A. Melo; Flávio V. Meirelles; Glaucius Oliva; Júlio C.M. Cascardo (pp. 115-123).

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein's structure was determined at 1.6 Å resolution and revealed a new topology for ACBP, containing five α-helices instead of four. α-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while α-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening.

Keywords: ACBP; Characterization; Crystal structure; Four-helix bundle; Moniliophthora perniciosa; and Witches' broom disease

Cell wall proteome of wheat roots under flooding stress using gel-based and LC MS/MS-based proteomics approaches by Fan-Jiang Kong; Atsushi Oyanagi; Setsuko Komatsu (pp. 124-136).

Cell wall proteins (CWPs) are important both for maintenance of cell structure and for responses to abiotic and biotic stresses. In this study, a destructive CWP purification procedure was adopted using wheat seedling roots and the purity of the CWP extract was confirmed by minimizing the activity of glucose-6-phosphate dehydrogenase, a cytoplasmic marker enzyme. To determine differentially expressed CWPs under flooding stress, gel-based proteomic and LC-MS/MS-based proteomic techniques were applied. Eighteen proteins were found to be significantly regulated in response to flood by gel-based proteomics and 15 proteins by LC MS/MS-based proteomics. Among the flooding down-regulated proteins, most were related to the glycolysis pathway and cell wall structure and modification. However, the most highly up-regulated proteins in response to flooding belong to the category of defense and disease response proteins. Among these differentially expressed proteins, only methionine synthase, β-1,3-glucanases, and β-glucosidase were consistently identified by both techniques. The down-regulation of these three proteins suggested that wheat seedlings respond to flooding stress by restricting cell growth to avoid energy consumption; by coordinating methionine assimilation and cell wall hydrolysis, CWPs played critical roles in flooding responsiveness.

Keywords: Abbreviations; CBB; Coomassie brilliant blue; CHAPS; 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonic acid; CWP; cell wall protein; 2-DE; two-dimensional polyacrylamide gel electrophoresis; DTT; dithiothreitol; G6PDH; glucose-6-phosphate dehydrogenase; IEF; isoelectric focusing; MM; molecular mass; nanoLC-MS/MS; nano liquid chromatography tandem mass spectrometry; pI; isoelectric point; PMSF; phenylmethylsulfonyl fluoride; PVPP; polyvinyl polypyrrolidoneCell wall; Flooding stress; LC-MS/MS; Proteomics; Wheat

Effect of detergent concentration on the thermal stability of a membrane protein: The case study of bacterial reaction center solubilized by N, N-dimethyldodecylamine- N-oxide by Gerardo Palazzo; Francesco Lopez; Antonia Mallardi (pp. 137-146).

We report on the response of reaction center (RC) from Rhodobacter sphaeroides (an archetype of membrane proteins) to the exposure at high temperature. The RCs have been solubilized in aqueous solution of the detergent N, N-dimethyldodecylamine- N-oxide (LDAO). Changes in the protein conformation have been probed by monitoring the variation in the absorbance of the bacteriochlorine cofactors and modification in the efficiency of energy transfer from tryptophans to cofactors and among the cofactors (through fluorescence measurements). The RC aggregation taking place at high temperature has been investigated by means of dynamic light scattering. Two experimental protocols have been used: (i) isothermal kinetics, in which the time evolution of RC after a sudden increase of the temperature is probed, and (ii) T-scans, in which the RCs are heated at constant rate. The analysis of the results coming from both the experiments indicates that the minimal kinetic scheme requires an equilibrium step and an irreversible process. The irreversible step is characterized by a activation energy of 205±14 kJ/mol and is independent from the detergent concentration. Since the temperature dependence of the aggregation rate was found to obey to the same law, the aggregation process is unfolding-limited. On the other hand, the equilibrium process between the native and a partially unfolded conformations was found to be strongly dependent on the detergent concentration. Increasing the LDAO content from 0.025 to 0.5 wt.% decreases the melting temperature from 49 to 42 °C. This corresponds to a sizeable (22 kJ/mol at 25 °C) destabilization of the native conformation induced by the detergent. The nature of the aggregates formed by the denatured RCs depends on the temperature. For temperature below 60 °C compact aggregates are formed while at 60 °C the clusters are less dense with a scaling relation between mass and size close to that expected for diffusion-limited aggregation. The aggregate final sizes formed at different temperatures indicate the presence of an even number of proteins suggesting that these clusters are formed by aggregation of dimers.

Keywords: Photosynthetic reaction center; Rhodobacter sphaeroides; Thermal denaturation; Detergent; Protein aggregation

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu by Hana Šanderová; Hana Tišerová; Ivan Barvík; Luděk Sojka; Jiří Jonák; Libor Krásný (pp. 147-155).

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.

Keywords: Thermostability; G protein; EF-Tu; G-domain; Bacillus

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation by Yuka Miyake; Satoshi Tsuzuki; Seiya Mochida; Tohru Fushiki; Kuniyo Inouye (pp. 156-165).

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation ( N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin–Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD- Myc(His)₆] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD- Myc(His)₆ exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.

Keywords: Abbreviations; endoH; endoglycosidase H; GPF; glycopeptidase F; HAI-1; hepatocyte growth factor activator inhibitor type 1; HGF; hepatocyte growth factor; MDCK cells; Madin–Darby canine kidney cells; MEM; minimum essential medium; r-EK; recombinant enterokinaseCatalytic domain; Matriptase; N; -glycosylation site; Protein folding; Zymogen activation

Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP by Hitomi Sawai; Shiro Yoshioka; Takeshi Uchida; Mamoru Hyodo; Yoshihiro Hayakawa; Koichiro Ishimori; Shigetoshi Aono (pp. 166-172).

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O₂, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O₂ sensing responsible for the regulation of the enzymatic activity.

Keywords: Abbreviations; HemDGC; heme-containing diguanylate cyclases; GCS(s); globin-coupled sensors; DGC(s); diguanylate cyclase(s); c-di-GMP; bis-(3′, 5′)-cyclic di-guanosine monophosphate; Hb(s); hemoglobin(s); HemAT; heme-containing aerotaxis transducer; MCP; methyl-accepting chemotaxis proteinDiguanylate cyclase; Oxygen sensor protein; Globin-coupled sensor; Heme; Bacterial second messenger

Concanavalin A aggregation and toxicity on cell cultures by Valeria Vetri; Rita Carrotta; Pasquale Picone; Marta Di Carlo; Valeria Militello (pp. 173-183).

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which–as a consequence–turns out to be non-toxic.

Keywords: Abbreviations; Con A; Concanavalin A; DLS; Dynamic light scattering; ThT; Thioflavin T; AFM; Atomic Force Microsopy; FTIR; Fourier transform infrared spectroscopy; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumConcanavalin A; Oligomers cytotoxicity; Amyloid fibril; Light scattering

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition by Akashi Ohtaki; Kensuke Murata; Yuichi Sato; Keiichi Noguchi; Hideyuki Miyatake; Naoshi Dohmae; Kazuhiro Yamada; Masafumi Yohda; Masfumi Odaka (pp. 184-192).

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/ Km values of 1.14±0.23 mM−¹s−¹, 4.54±0.09 mM−¹s−¹, 0.087±0.02 mM−¹s−¹ and 153.5±7.1 mM−¹s−¹, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser- cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.

Keywords: Abbreviations; AS; amidase signature; RhAmidase; Rhodococcus; sp. N-771 amidase; S195A; mutant RhAmidase in which Ser195 is replaced by Ala; MAE2; Bradyrhizobium japonicum; malonamidase; Pam; Stenotrophomonas maltophilica; peptide amidaseAmidase; Amidase signature family; Rhodococcus; sp. N-771; Crystal structure; Substrate specificity

Effect of pressure on the secondary structure of coiled coil peptide GCN4-p1 by Hiroshi Imamura; Yasuhiro Isogai; Takahiro Takekiyo; Minoru Kato (pp. 193-198).

It has recently been demonstrated that pressure induces folding of the α-helix of an alanine-based peptide (AK20), which is a monomer in water (Imamura and Kato, Proteins 2009;76:911–918). The present study focused on a coiled coil peptide GCN4-p1, the α-helices of which associate via a hydrophobic core, to examine whether the pressure stability of the α-helices depends on the hydrophobic core. Fourier transform infrared spectroscopy was used to investigate the effect of pressure on the secondary structures of GCN4-p1. The infrared spectra of GCN4-p1 shows the two amide I' peaks at ∼1650 and ∼1630 cm−¹ stemming from the solvent-inaccessible α-helix and the solvent-accessible α-helix, respectively. The intensities of both the peaks increase with increasing pressure, whereas they decrease with increasing temperature. This indicates that pressure induces both the α-helices of GCN4-p1 to fold. The present result suggests that the positive volume change upon unfolding of an α-helix is a common characteristic of peptides. The pressure-induced stabilization of the α-helices is discussed in comparison with the pressure denaturation of proteins.

Keywords: Pressure effect; Coiled coil; GCN4-p1; Infrared spectroscopy; Alpha-helix; CD spectroscopy

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase by Paul V. Attwood; Katrin Ludwig; Klaus Bergander; Paul G. Besant; Abdussalam Adina-Zada; Josef Krieglstein; Susanne Klumpp (pp. 199-205).

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

Keywords: Protein histidine phosphatase; Phosphohistidine; Histidine kinase; Histone H4

Relevance of glycine and cysteine residues as well as N- and C-terminals for the activity of protein histidine phosphatase by Susanne Klumpp; Nien Tze Ma; Nicole Bäumer; Gunther Bechmann; Josef Krieglstein (pp. 206-211).

There is increasing evidence that reversible phosphorylation of histidine residues regulates numerous important cellular processes. The first protein histidine phosphatase (PHP) from vertebrates was discovered just recently. Here, we report on amino acids and domains essential for activity of PHP. Point mutations of conserved residues and deletions of the N- and C-termini of PHP were analyzed using [³²P-his]ATP-citrate lyase as a substrate. Individual or joint replacement of all cysteine residues by alanine did not affect PHP activity. Deletion of 9 N-terminal amino acids resulted in inactive PHP. Furthermore, only 4 C-terminal residues could be deleted without losing PHP activity. Single or multiple mutations of the glycine-rich domain (Gly⁷⁵, Gly⁷⁷) of a putative nucleotide binding site of PHP (GxGxxG/S) caused inactivation of PHP. Wildtype PHP could be labeled with [α-³²P]ATP. Such radiolabeling was not detectable for catalytically inactive PHP-G75A and PHP-G77A. These data suggest further studies on the interaction between PHP and ATP.

Keywords: Abbreviations; PHP; protein histidine phosphatase; ACL; ATP-citrate lyase; NDPK; nucleoside diphosphate kinaseProtein histidine phosphatase; Histidine; Glycine; ATP; Site-directed mutagenesis

A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3 by Toshana L. Foster; Philip R. Tedbury; Arwen R. Pearson; Mark Harris (pp. 212-222).

Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

Keywords: Abbreviations; HCV; Hepatitis C virus; NS; non-structural; JFH-1; Japanese fulminant hepatitis C virus genotype 2a isolate; WT; wild-type; GdnHCl; guanidine hydrochloride; DTT; dithiothreitol; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CD; circular dichroismHepatitis C virus; NS2-3 autoprotease; Mutagenesis; Refolding; Tryptophan fluorescence; Acrylamide quenching

Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH by Basir Ahmad; Julia Winkelmann; Bruno Tiribilli; Fabrizio Chiti (pp. 223-234).

Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimer's disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. Interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive β-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0–3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction.

Keywords: Amyloid-like oligomer; Alkaline pH; Congo red; HypF-N; Premolten globule

Contributions of active site residues to cofactor binding and catalysis of 3α-hydroxysteroid dehydrogenase/carbonyl reductase by Yi-Hsun Chang; Chau-Zen Wang; Chien-Chih Chiu; Lea-Yea Chuang; Chi-Ching Hwang (pp. 235-241).

3α-Hydroxysteroid dehydrogenase/carbonyl reductase reversely catalyzes the oxidation of androsterone with NAD⁺ to form androstanedione and NADH. In this study, we investigated the function of active site residues N86, Y155, and K159 in NADH binding and catalysis in the reduction of androstanedione, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching, and anisotropy measurements. The N86A, Y155F, and K159A mutant enzymes decreased the catalytic constant by 37- to 220-fold and increased the dissociation constant by 3- to 75-fold, respectively. Binding of NADH with wild-type and mutant enzymes caused different levels of fluorescence resonance energy transfer, implying a different orientation of nicotinamide ring versus W173. In addition, the enzyme-bound NADH decreased the fluorescence anisotropy value in the order WT>N86A>Y155F>K159A, indicating an increase in the mobility of the bound NADH for the mutants. Data suggest that hydrogen bonding with the hydroxyl group of nicotinamide ribose by K159 and Y155 is important to maintain the orientation of NADH and contributes greatly to the transition-state binding energy to facilitate the catalysis. N86 is important for stabilizing the position of K159. Substitution of alanine for N86 has a minor effect on NADH binding through K159, resulting in a slight increase in the mobility of the bound NADH and decreases in affinity and catalytic constant.

Keywords: Abbreviations; 3α-HSD/CR; 3α-hydroxysteroid dehydrogenase/carbonyl reductase; SDR; short chain dehydrogenase/reductase; FRET; fluorescence resonance energy transferCofactor binding; Enzyme catalysis; Mutagenesis; NADH; Hydroxysteroid dehydrogenase

Sections

Personal tools

BBA - Proteins and Proteomics (v.1804, #1)

Sections

Personal tools

Local resources

BBA - Proteins and Proteomics (v.1804, #1)