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BBA - Proteins and Proteomics (v.1764, #10)

Editorial Board (pp. ii).

Picosecond dynamics of T and R forms of aspartate transcarbamylase: A neutron scattering study by J.-M. Zanotti; G. Hervé; M.-C. Bellissent-Funel (pp. 1527-1535).

E. coli aspartate transcarbamylase (ATCase) is a 310 kDa allosteric enzyme which catalyses the first committed step in pyrimidine biosynthesis. The binding of its substrates, carbamylphosphate and aspartate, induces significant conformational changes. This enzyme shows homotropic cooperative interactions between the catalytic sites for the binding of aspartate. This property is explained by a quaternary structure transition from T state (aspartate low affinity) to R state (aspartate high affinity) accompanied by a 5% increase of radius of gyration of ATCase. The same quaternary structure change is observed upon binding of the bisubstrate analogue PALA ( N-(phosphonacetyl)-l-aspartate. Owing to the large incoherent neutron scattering cross-section of the hydrogen atom and the abundance of this element in proteins, inelastic neutron scattering gives a global view of protein dynamics as sensed via the individual motions of its hydrogen atoms. We present neutron scattering results of the local dynamics (few angstroms), at short time (few tens of picoseconds), of ATCase in T and R forms. Compared to the T form, we observe an increased mobility of the protein in the R form that we associate to an increase of accessible surface area to the solvent. Beyond this specific result, this highlights the key role of the accessible surface area (ASA) in dynamic contribution to inelastic neutron data in the picosecond time scale. In particular, we want to stress out (i) that a difference at the picosecond time scale does not allow to conclude to a difference in the dynamics at a longer time scale and to address whether the T state is looser than the R state (ii) how challenging is, any comparison in terms of general dynamics (tense or relaxed) between dynamic values deduced from experimental neutron data on proteins with different sequences and therefore ASA. This caveat holds particularly when comparing dynamics of a mesophile with the corresponding extremophile.

Keywords: Aspartate transcarbamylase; Allosteric enzyme; Regulation; Dynamic; Inelastic neutron scattering

Proteome analysis of myocardial tissue following ischemia and reperfusion—Effects of complement inhibition by Michael Buerke; Hansjörg Schwertz; Tina Längin; Ute Buerke; Roland Prondzinsky; Herbert Platsch; Joachim Richert; Sabine Bomm; Martin Schmidt; Heinz Hillen; Stephan Lindemann; Gottfried Blaschke; Ursula Müller-Werdan; Karl Werdan (pp. 1536-1545).

Myocardial ischemia–reperfusion injury can be related to complement activation with generation of chemotactic mediators, release of cytokines, leukocyte accumulation, and subsequent severe tissue injury. In this regard, activation of transcription factors (i.e., NFκB) and de novo protein synthesis or inflammatory protein degradation seems to play an important role. In the present study, we analyzed the cardiac protein expression following myocardial ischemia (60 min) and reperfusion (180 min) in a rabbit model utilizing two-dimensional electrophoresis and nanoHPLC/ESI-MS/MS for biochemical protein identification. To achieve cardioprotective effects, we used a novel highly selective small molecule C1s inhibitor administered 5 min prior to reperfusion. The reduction of myocardial injury was observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (65.2±3 vs. 38.5±3 U/g protein after 3 h of reperfusion, P<0.05). With proteome analysis we were able to detect 509±21 protein spots on the gels of the 3 groups. A pattern of 480 spots with identical positions was found on every gel of myocardial tissue of sham animals, vehicle and C1s-INH-248-treated animals. We analyzed 11 spots, which were identified by mass spectrometry: Superoxide dismutase, α-crystallin-chain-B, mitochondrial stress protein, Mn SOD, ATP synthase A chain heart isoform, creatine kinase, and troponin T. All of these proteins were significantly decreased in the vehicle group when we compared to sham-treated animals. Treatment with C1s-INH-248 preserved levels of these proteins. Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex archives cardio-protection by altering and preserving different anti-inflammatory and cytoprotective cascades.

Keywords: Abbreviations; C1s-INH-248; synthetic small molecule inhibitor of C1s; CK; creatine kinase; C5b-9 or MAC; membrane attack complex; U; unit; MS; mass spectrometry; 2D; 2 dimensional; IP; isoelectric pointProteomics; Two-dimensional electrophoresis; Mass spectrometry; Small molecule inhibitor of C1s; Complement; Ischemia–reperfusion injury; Neutrophil

Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration by Allison J. Richard; Chin-Chi Liu; Alexandra L. Klinger; Matthew J. Todd; Tara M. Mezzasalma; Vince J. LiCata (pp. 1546-1552).

The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 °C, from 40 to 62 °C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH₄⁺>Na⁺>Li⁺>K⁺. Of the divalent cations tested: Mg⁺² and Mn⁺² significantly stabilize the protein, while Ni⁺² dramatically destabilizes the protein. Stability measurements performed in combined Mg⁺² plus Na⁺ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.

Keywords: Pol I; Thermofluor; Denaturation; Unfolding; Cationic stabilization

Dynamic and coordinating domain motions in the active subunits of the F₁-ATPase molecular motor by Ming S. Liu; B.D. Todd; Richard J. Sadus (pp. 1553-1560).

F₁-ATPase is a rotary molecular motor crucial for various cellular functions. In F₁-ATPase, the rotation of the γδε subunits against the hexameric α₃β₃ subunits is highly coordinative, driven by ATP hydrolysis and structural changes at three β subunits. However, the dynamical and coordinating structural transitions in the β subunits are not fully understood at the molecular level. Here we examine structural transitions and domain motions in the active subunits of F₁-ATPase via dynamical domain analysis of the α₃β₃γδε complex. The domain movement and hinge axes and bending residues have been identified and determined for various conformational changes of the β-subunits. P-loop and the ATP-binding pocket are for the first time found to play essential mechanical functions additional to the catalytic roles. The cooperative conformational changes pertaining to the rotary mechanism of F₁-ATPase appears to be more complex than Boyer's ‘bi-site’ activity. These findings provide unique molecular insights into dynamic and cooperative domain motions in F₁-ATPase.

Keywords: F; ₁; -ATPase; Molecular motor; Dynamic domain; Coarse-grained dynamics; Structural transition; Conformational cooperativity

Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein by Takeshi Yamazaki; Chisa Matsuoka; Miho Gendou; Shunsuke Izumi; Dong Zhao; Irina Artemenko; Colin R. Jefcoate; Shiro Kominami (pp. 1561-1567).

Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40–285 and 55–285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39–40 and 54–55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.

Keywords: Steroidogenesis regulation; Adrenal gland; Steroidogenic acute regulatory protein; Mitochondrial processing peptidase

Structural characterization of an unusually stable cyclic peptide, kalata B2 from Oldenlandia affinis by Sudarslal Sadasivan Nair; Julija Romanuka; Martin Billeter; Lars Skjeldal; Mark R. Emmett; Carol L. Nilsson; Alan G. Marshall (pp. 1568-1576).

Kalata peptides are isolated from an African medicinal plant, Oldenlandia affinis, an aqueous decoction of which can be ingested to accelerate uterine contraction during childbirth. The closely packed disulfide core of kalata peptides confers unusual stability against thermal, chemical, and enzymatic degradation. The molecular arrangement may hamper NMR-assisted disulfide connectivity assignment. We have combined NMR with high-resolution mass spectrometry (MS) and MS/MS of native and chemically derivatized kalata B2 to determine its amino acid sequence and disulfide connectivity. Infrared multiphoton dissociation establishes the disulfide bond linkages in kalata B2 as I–IV, II–V and III–VI.

Keywords: Disulfide bond; Cyclic peptide; NMR spectroscopy; Mass spectrometry

A novel type of lysine oxidase:l-lysine-ε-oxidase by Daniel Gómez; Patricia Lucas-Elío; Antonio Sanchez-Amat; Francisco Solano (pp. 1577-1585).

The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas–Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193–203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493–2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of freel-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H₂O_2, namely lysine-α-oxidase (LαO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-ε-oxidase ( lod for the gene) instead of marinocine. Lod shows high stereospecificity for freel-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by β-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site.

Keywords: Abbreviations; AAN; aminoacetonitrile; βAPN; β-aminopropionitrile; LAO; l; -amino acid oxidase; LαO; Lysine-α-oxidase; Lat; Lysine-ε-aminotransferase; Lod; Marinocine (Lysine-ε-oxidase); Lox; Lysyl oxidase; LTQ; Lysyl-Tyrosyl-Quinone; SDH; saccharopine dehydrogenase; SSAO; semicarbazide sensitive amino oxidase; TDQC; tyrosine-derived quinone cofactors; TLC; Thin layer chromatographyl; -lysine; Amino acid oxidases; Deamination; Antibacterial activity; Hydrogen peroxide

Fluorescence contributions of the individual Trp residues in goat α-lactalbumin by Ann Vanhooren; Eszter Illyes; Zsuzsa Majer; Ignace Hanssens (pp. 1586-1591).

Goat α-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.

Keywords: Alpha-lactalbumin; Trp-Phe mutant; Differential scanning calorimetry; Tryptophan fluorescence; Disulfide bond; Photolysis

BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na⁺ channel toxin BcIII by Joacir Stolarz Oliveira; André Junqueira Zaharenko; Wilson Alves Ferreira Jr.; Katsuhiro Konno; Cláudio Saburo Shida; Michael Richardson; Aline Duarte Lúcio; Paulo Sérgio Lacerda Beirão; José Carlos de Freitas (pp. 1592-1600).

Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 μg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD₅₀=219 μg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of β-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na⁺ channel effector and BcIV is inactive over Na⁺ currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.

Keywords: Abbreviations; amu; atomic mass unit; Arg-EP; arginylendopeptidase; ASIC; acid-sensing ion channel; BcIII and BcIV; Bunodosoma caissarum; toxins III and IV, respectively; CAPs; compound action potentials; CD; circular dichroism; CH; ₃; CN; acetonitrile; DTT; dithiothreitol; HERG; human ether-a-gogo related gene potassium channel; Kv; voltage-gated potassium channel; Lys-EP; lysylendopeptidase; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ESI-Q-TOF MS/MS; electrospray ionization quadrupole tandem mass spectrometry; Nav; voltage-gated sodium channel; rp-HPLC; reversed-phase high performance liquid chromatography; TFA; trifluoroacetic acid Bunodosoma caissarum; BcIII; BcIV; Circular dichroism; MS/MS spectrometry; Sea anemone; Molecular modeling

Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains by Gunnar Höst; Lars-Göran Mårtensson; Bengt-Harald Jonsson (pp. 1601-1606).

The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is >500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

Keywords: Carbonic anhydrase; Hydrolysis; Specificity; Mutagenesis; Protein engineering; Rational design

Proteomic inventory of “Anchorless�? proteins on the colon adenocarcinoma cell surface by Harold Tjalsma; Wendy Pluk; Lambert P. van den Heuvel; Wilbert H.M. Peters; Rian Roelofs; Dorine W. Swinkels (pp. 1607-1617).

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several “non-membrane�? proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called “anchorless�? surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble “surface�? proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of “intracellular�? and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of “anchorless�? proteins to the surface of colon tumor cells.

Keywords: Cell surface proteome; Shotgun proteomics; Colon cancer; SW480 cell

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells by Holger Cynis; Stephan Schilling; Mandy Bodnár; Torsten Hoffmann; Ulrich Heiser; Takaomi C. Saido; Hans-Ulrich Demuth (pp. 1618-1625).

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Aβ(N3E-42) and Aβ(N3Q-42) was investigated. These amyloid-β peptides were expressed as fusion proteins with either the pre–pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Aβ(N3Q-42) after β- and γ-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Aβ-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.

Keywords: Abbreviations; Aβ; amyloid β-peptide; APP; amyloid precursor protein; β-CTF; β-C-terminal fragment of APP; DMEM; Dulbecco's modified eagle medium; GnRH; gonadotropin releasing hormone; HRP; horseradish peroxidase; pGlu; pyroglutamic acid; QC; glutaminyl cyclase; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; TMB; tetramethylbenzidine; mTRH; murine thyrotropin releasing hormoneGlutaminyl Cyclase; Pyroglutamic acid; Inhibition; Amyloid-β; Alzheimer's disease

Limitations in quantitation of the biomarker CCL18 in Gaucher disease blood samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry by Mariëlle J. van Breemen; Boris Bleijlevens; Chris G. de Koster; Johannes M.F.G. Aerts (pp. 1626-1632).

SELDI-TOF MS assisted the discovery of the chemokine CCL18/PARC as plasma biomarker for pathological storage cells in Gaucher disease patients. Prognostic elevation of CCL18 in blood of Gaucher patients has been confirmed by ELISA. Given its low molecular mass, positive charge, and relatively high abundance, CCL18 seems a particular attractive protein for SELDI-TOF based quantitation. Therefore, we determined CCL18 levels in plasma using SELDI-TOF MS and ELISA, in parallel. CCL18 levels in some blood samples were significantly underestimated when determined by SELDI-TOF MS. Spiking of recombinant CCL18 indicated that its detection by SELDI-TOF MS is strongly determined by the nature of the sample, even markedly varying between samples obtained from one donor at different time points. Independent of the total CCL18 concentration in blood only 1–10% of the chemokine bound to the ProteinChip® Array. Even when comparable amounts of CCL18 from distinct samples were bound to the ProteinChip® Array, diverse peak intensities could be observed. Thus, limited binding capacity and sample-dependent suppression of CCL18 ionization contribute significantly to the final peak intensity. In conclusion, SELDI-TOF MS offers no reliable procedure to quantitatively monitor CCL18 levels in blood and thus cannot be applied in evaluation of disease status of Gaucher patients.

Keywords: Abbreviations; ERT; enzyme replacement therapy; GD; Gaucher disease; rhCCL18; recombinant human CCL18; SRT; substrate reduction therapyBiomarker; CCL18/PARC; Gaucher disease; Plasma; Serum; Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

Contribution of W229 to the transglycosylation activity of 4-α-glucanotransferase from Pyrococcus furiosus by Shuang-Yan Tang; Sung-Jae Yang; Hyunju Cha; Eui-Jeon Woo; Cheonseok Park; Kwan-Hwa Park (pp. 1633-1638).

A W229H mutant of 4-α-glucanotransferase (4-α-GTase) from Pyrococcus furiosus was constructed and its catalytic properties were studied to investigate the role of W229 in the catalytic specificities of the enzyme. Various activities and kinetic parameters were determined for the wild-type and W229H mutant enzymes. The transglycosylation factor and transglycosylation activity of the mutant enzyme markedly decreased, but its hydrolysis activity was scarcely affected. It was discovered that the k_cat/ K_m value of transglycosylation activity significantly decreased to about 15% of that of the wild type, while k_cat/ K_m value of hydrolysis activity changed little for the mutant enzyme. The hydrophobicity of W229 was thought to be critical to the transglycosylation activity of the enzyme based on the enzyme's modeled tertiary structures.

Keywords: 4-α-glucanotransferase (4-α-GTase); Site-directed mutagenesis; Transglycosylation activity; Hydrolysis activity; Hydrophobicity

Comparison of the internal dynamics of globular proteins in the microcrystalline and rehydrated lyophilized states by Alexey Krushelnitsky; Yuri Gogolev; Ralph Golbik; Frederick Dahlquist; Detlef Reichert (pp. 1639-1645).

Natural abundance solid-state¹³C-NMR spin-lattice relaxation experiments in the laboratory ( T₁) and off-resonance rotating ( T_1�?) frames were applied for qualitative comparison of the internal molecular dynamics of barstar, hen egg white lysozyme and bacteriophage T4 lysozyme in both the microcrystalline and the rehydrated (water content is 50% of the protein mass) lyophilized states. The microcrystalline state of proteins provides a better spectral resolution; however, less is known about the local structure and dynamics in the different states. We found by visual comparison of both T₁ and T_1�? relaxation decays of various resonance bands of the CPMAS spectra that within the ns–μs range of correlation times there is no appreciable difference in the internal dynamics between rehydrated lyophilized and crystalline states for all three proteins tested. This suggests that the internal conformational dynamics depends weakly if at all on inter-protein interactions in the solid state. Hence, physical properties of globular proteins in a fully hydrated solid state seem to be similar to those in solution. This result at least partly removes concerns about biological relevance of studies of globular proteins in the solid state.

Keywords: Proteins; Dynamics; Solid-state NMR; Microcrystals; Powders

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BBA - Proteins and Proteomics (v.1764, #10)