Side-chain Motions Research Articles

Subglass and Glass Transitions of Poly(di-n-alkylitaconate)s with Various Side-Chain Lengths: Dielectric Relaxation Investigation

Dielectric relaxation measurements were performed on a series of poly(di-n-alkylitaconate)s with various side-chain lengths. The experimental data were analyzed by adjustment with one or the sum of several Havriliak−Negami functions. They demonstrated the existence of several relaxation processes with significant dielectric relaxation strengths. The comparison of the results obtained by increasing the length of the side groups permitted an assignment of the γ, βfast, and βslow secondary relaxations to motions of the alkyl side chains, ester group located after the CH2 unit, and ester group directly attached to the main chain, respectively. In poly(di-n-alkylitaconate)s with six carbon atoms at least in the alkyl part, the existence of an additional low-temperature glass transition αL was clearly established. The αL-process replaces the βfast-process observed in the lower derivatives. It is located in quite the same temperature and frequency ranges and exhibits a non-Arrhenius behavior. It is likely relate...

Pulsed NMR Study of Network Formation in the Course of Bulk Polymerization of Methyl Acrylate

The proton spin-spin relaxation time (T2) during the bulk polymerization of methyl acrylate was measured as a function of the reaction time at various temperatures. Three kinds of T2 (T2L (long), T2S (short) and T2M (intermediate)) were obtained as the polymerization proceeded. The fraction of T2S (FS) increased sigmoidally at a certain reaction time, while that of T2L (FL) decreased reciprocally. The former corresponded to the amount of a polymer whose molecular weight was sufficiently high enough to cause a tight entanglement that produced a transient network structure; the latter reflected a decrease in the mixture of the monomer and the low molecular weight of the polymer. T2M is considered to arise from a relatively mobile region of the entanglement. The relationship between the fractions of T2S + T2M and the polymer yield was found to be linear, which led us to monitor the polymer yield in real time during the polymerization in a non-distractive manner. 13C DD (dipolar decoupling)/MAS (magic angle spinning) NMR spectra were also measured to monitor the polymerization process in terms of the molecular motions between the main chain and the side chain in the formation of a network structure. The 13C DD/MAS NMR spectra show that the side chain motion became restricted as well as the main chain when the "Trommosdorff effect" (gel effect) was observed, and a part of the monomers were trapped in the network structure.

Time−Temperature Superposition for Viscoelastic Properties of Regioregular Poly(3-hexylthiophene) Films

Shear moduli were determined for chemically polymerized and solvent cast regioregular poly(3-hexylthiophene) films, using thickness shear mode acoustic wave resonators. The results are strikingly different to those for electropolymerized regiorandom poly(3-hexylthiophene) films. The time scale of the measurement was varied directly by use of higher harmonics of the acoustic wave resonator and indirectly via temperature. The significant variations in shear modulus with effective time scale can be "normalized"onto a stress master relaxation curve by using the concept of time-temperature superposition; this is the first time this has been demonstrated for electroactive films. The shift factors required to effect this normalization do not follow the classical Williams-Landel-Ferry (WLF) equation developed for long-range backbone motions of bulk polymers. Instead, they follow an Arrhenius-like behavior, commonly used to describe secondary motions of polymer side-chains. The activation enthalpy associated with this is independent of applied potential, is the same as for as cast (undoped) films, and is similar to that for rotation about a carbon-carbon single bond. These all point to the hexyl side-chains as the origins of the observed phenomena, consistent with the "melting point" separating two temperature-dependent phases and with the different molecular packing arrangements that would necessarily apply to regioregular and regiorandom materials.

Penetratin-Membrane Association: W48/R52/W56 Shield the Peptide from the Aqueous Phase

Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a π-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate.

The Crystal Structure of Guinea Pig 11β-Hydroxysteroid Dehydrogenase Type 1 Provides a Model for Enzyme-Lipid Bilayer Interactions

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulin secretion. This reaction is carried out by the NADPH-dependent type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11beta-HSD1. This variant was determined in complex with NADP at 2.5 A resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11beta-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.

Some aspects of structural transformations taking place in organic mass of Ukrainian coals during heating. Part 1. Study of structural transformations when heating coals of different caking capacity

The structure of the plastic layer in coals of different caking capacity was studied using the X-ray method and the influence of the plastic layer structure on the mechanism of coke formation was determined. It is shown that the transportation of the plastic mass in the gas-saturated zone of the plastic layer has a decisive influence on the decrease in volume of the coal charge and on obtaining a denser residue during carbonization. When heating poorly caking and non-caking coals an increase in the charge volume takes place. Using the quantitative X-ray phase analysis an ordered phase amount ( C cryst) was found in solid residues of carbonization and the parameters d 002, L c and L a were determined. It is shown that the gas-saturated zone is generated in the plastic layer only when structural transformations in coals take place at the pre-plastic stage without changing the ratio between the number of carbon atoms in the ordered and non-ordered phases. The transition of well caking coal into the plastic state is accompanied by the activation of segmental and conformal movement of side chains and separate macromolecular fragments of the coal organic mass. In the course of grain swelling of non-caking coal its crystallites degrade. The conclusions drawn on the basis of X-ray studies were confirmed by the EPR-spectroscopy of residues corresponding to plastic zones in coals of different caking capacity.

Phylogeny of protein-folding trajectories reveals a unique pathway to native structure

To scrutinize how a protein folds at atomic resolution, we performed 200 molecular dynamics simulations (each of 50 ns) of the miniprotein Trp-cage on the computational grid. Within the trajectories, 58 folding and 31 unfolding events were identified and subjected to extensive comparison and classification. Based on an analogy with biological sequences, the folding and unfolding trajectories (arrays of sequential snapshots of structures) were aligned by dynamic programming allowing gaps. A phylogenetic tree derived from the alignments revealed four distinct groups of the trajectories, characterized by the Trp side-chain motions and the main-chain motions. It was found that only one group attained the native structure and that the other three led to pseudonative structures having the correct main-chain trace but different nonnative Trp side-chain rotamers, indicating that those four folded structures were each attained through a unique folding pathway.

The impact of protein flexibility on protein–protein docking

Accounting for protein flexibility in protein-protein docking algorithms is challenging, and most algorithms therefore treat proteins as rigid bodies or permit side-chain motion only. While the consequences are obvious when there are large conformational changes upon binding, the situation is less clear for the modest conformational changes that occur upon formation of most protein-protein complexes. We have therefore studied the impact of local protein flexibility on protein-protein association by means of rigid body and torsion angle dynamics simulation. The binding of barnase and barstar was chosen as a model system for this study, because the complexation of these 2 proteins is well-characterized experimentally, and the conformational changes accompanying binding are modest. On the side-chain level, we show that the orientation of particular residues at the interface (so-called hotspot residues) have a crucial influence on the way contacts are established during docking from short protein separations of approximately 5 A. However, side-chain torsion angle dynamics simulations did not result in satisfactory docking of the proteins when using the unbound protein structures. This can be explained by our observations that, on the backbone level, even small (2 A) local loop deformations affect the dynamics of contact formation upon docking. Complementary shape-based docking calculations confirm this result, which indicates that both side-chain and backbone levels of flexibility influence short-range protein-protein association and should be treated simultaneously for atomic-detail computational docking of proteins.

A Multifrequency Electron Spin Resonance Study of T4 Lysozyme Dynamics Using the Slowly Relaxing Local Structure Model

Electron spin resonance (ESR) spectra were obtained at 250 and 9 GHz for nitroxide-labeled mutants of the protein T4 lysozyme in aqueous solution over a range of temperatures from 2 to 37.5 °C. Two mutants labeled at sites 72 and 131 were studied and compared. The mutant sites are solvent exposed and free of tertiary interactions with other side chains, but the former is at the center of a 5 turn helix, whereas the latter site is on a small two and a half turn helix. The 250 GHz ESR spectra, because of their “fast time scale”, are rather insensitive to the slow overall tumbling motion of the protein. Thus, they are qualitatively different for the two mutants, implying that there are different local dynamics at the two sites. The 9 GHz spectra, which are significantly affected by the overall tumbling and are less sensitive to the internal dynamics, do not show such marked differences between the two sites. The 250 and 9 GHz spectra for each mutant and temperature were simultaneously fit to the slowly relaxing local structure (SRLS) model for slow-motional ESR, using newly developed software. The SRLS model explicitly accounts for the overall tumbling of the protein and the internal modes of motion, which include the motion of the nitroxide side chain (expected to be the same for both mutants) and backbone fluctuations. Very good simultaneous fits are obtained. Whereas two conformers (or spectral components) are typically detected at the lower temperatures, only a single component is observed at the higher temperatures. The significant differences in the high-frequency spectra for the two mutants are readily attributed mainly to a difference in their respective local ordering. That is, site 72 exhibits significantly greater local ordering than does site 131, which is expected from the greater rigidity of the larger helix on which the 72 site is located. The results of this multifrequency study are compared with a previous 9 GHz study. A description of the application of the SRLS model in such a multifrequency study is provided.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the α-helical surface loop, the so-called ‘lid’ of HLL comprised of residues 86–93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the ‘open’ conformation in triacetin, with the α-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.

Time-Resolved CIDNP Study of Native-State Bovine and Human α-Lactalbumins

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of two aromatic dyes (2,2‘-dipyridyl, DP, and flavin mononucleotide, FMN) with bovine and human α-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. With DP as the photosensitizer, polarization in BLA is observed for the protons of Trp118, His68, Tyr18, and Tyr103. The latter is not manifested in the CIDNP spectra obtained with FMN. This threshold effect on CIDNP formation indicates that accessibility is a combined property of both the residue and the dye. The nuclear spin−lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T1 = 60 μs for H2,4,7 of Trp118, T1 = 53 μs for H3,5 of Tyr103, T1 = 16 μs for H3,5 of Tyr18, and T1 = 10 μs for the H2 and H4 of His68. The correlation times for the side chain motion as determined from the T1 of the radicals are correlated with the accessibility of the side chains in the intact protein.

Altered hydrogen bonding of Arg82 during the proton pump cycle of bacteriorhodopsin: a low-temperature polarized FTIR spectroscopic study.

Light-driven proton transport in bacteriorhodopsin (BR) is achieved by dynamic rearrangement of the hydrogen-bonding network inside the membrane protein. Arg82 is located between the Schiff base region and proton release group, and has a major influence on the pK(a) values of these groups. It is believed that Arg82 changes its hydrogen-bonding acceptors during the pump cycle of BR, stages of which are correlated with proton movement along the transport pathway. In this study, we compare low-temperature polarized FTIR spectra of [eta(1,2)-(15)N]arginine-labeled BR in the 2750-2000 cm(-1) region with those of unlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, (15)N-shifted modes were found at 2292 (-)/2266 (+) cm(-1) and at 2579 (-)/2567 (+) cm(-1). The former corresponds to strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-D stretches probably originate from Arg82, the former oriented toward water 406 and the latter toward the extracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization. This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no (15)N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is not significantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolar orientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular to parallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 side chain from the Schiff base region to the extracellular proton release group. Additional bands corresponding to weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes in hydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.

Time-Resolved Fluorescence Anisotropy in Assessing Side-Chain and Segmental Motions in Polyamines Entrapped in Sol−Gel Derived Silica

The side-chain and backbone dynamics of two model polyamines, polylysine (PL) and poly(allylamine) (PAM), were examined with time-resolved fluorescence anisotropy (TRFA) in aqueous solution and when the polyamines were entrapped into sol−gel derived silica. Both polyamines were ionically labeled with fluorescein, causing the rotational characteristics of the probe to be interconnected to the dynamics of the polyamine chain. TRFA studies of the probe−polyamine complex could be fit to two rotational components reflecting motions of the side chains (ps component) and short segments (ns component), respectively. The rate and amplitude of these motions were reproducibly higher for PL than PAM, indicative of a higher conformational flexibility in PL relative to PAM. This result was supported by molecular mechanics optimizations, which showed a much larger variance in the distance between adjacent amino groups in PL relative to PAM, consistent with more degrees of freedom in the more dynamic polyamine. When entrapped into sodium silicate (SS)-derived hydrogels, PL unexpectedly showed a high degree of segmental flexibility, while PAM experienced a significant damping of all detectable motions, accompanied by a large increase in the residual anisotropy. Since the random coil of PL can be considered as a model of flexible or denatured proteins with a high affinity toward the silica surface, we would expect the presence of segmental motions in such proteins when entrapped into a SS network, even if these are bound to the silica surface, in agreement with previous studies of such proteins entrapped in sol−gel glasses. On the other hand, PAM provides a useful model of more rigid proteins, such as antibodies, which show significant losses in dynamic motion upon entrapment in silica. The results show that the dynamics of proteins entrapped in silica materials must be viewed with caution, as it is possible to have significant dynamic motion even when proteins interact with the matrix.

Critical role of magnesium ions in DNA polymerase beta's closing and active site assembly.

To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerase mechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles in the conformational transitions between closed and open states and assembly/disassembly of the active site of polymerase beta/DNA complexes before and after the chemical reaction of nucleotide incorporation. Using dynamics simulations, we find that closing before chemical reaction requires both divalent metal ions in the active site while opening after the chemical reaction is triggered by release of the catalytic metal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalytic ion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group through the nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg(2+) and Na(+) ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ion itself is not a rate-limiting conformational or overall step. However, geometric adjustments associated with functional ions and proper positioning in the active site, including subtle but systematic motions of protein side chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelity mechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotide approaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding and catalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptions of polymerase pathways and underscores the importance of the delicate conformational events both before and after the chemical reaction to polymerase efficiency and fidelity mechanisms.

The origin of protein sidechain order parameter distributions.

Previous work by Wand et al. (Nature 2001, 411, 501-504) showed that the NMR order parameters characterizing the amplitude of motion of protein side chains seemed to form a multimodal distribution. At the time, no detailed explanation of this at the molecular level was offered, yet three "classes" of motion were inferred. We have analyzed a larger published data set and found that, although the distribution is multimodal, the evidence for three classes is weak. More significantly, we have been able to provide a simple physical explanation for the distributions based on the results of molecular dynamics simulations. This result will aid in the interpretation of data from NMR dynamics experiments.

Side chain dynamics monitored by 13C-13C cross-relaxation.

A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves. The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy.

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.

Structural effects of radiation damage and its potential for phasing

A detailed analysis of radiation-damage-induced structural and intensity changes is presented on the model protein thaumatin. Changes in reflection intensities induced by irradiation display a parabolic character. The most pronounced structural changes observed were disulfide-bond breakage and associated main-chain and side-chain movements as well as decarboxylation of aspartate and glutamate residues. The structural changes induced on the sulfur atoms were successfully used to obtain high-quality phase estimates via an RIP procedure. Results obtained with ACORN suggest that the contribution originating from the partial structure may play an important role in phasing even at less than atomic resolution.

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA.

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.

The Roles of Glu186 and Glu380 in the Catalytic Reaction of Soybean β-Amylase

It has previously been suggested that the glutamic acid residues Glu186 and Glu380 of soybean β-amylase play critical roles as a general acid and a general base catalyst, respectively. In order to confirm the roles of Glu186 and Glu380, each residue was mutated to a glutamine residue and the crystal structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose) complexes were determined at resolutions of 1.6 Å and 1.9 Å, respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased activity relative to that of the wild-type enzyme. The crystal structure of the E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at subsites −2 to +3. Two maltose molecules bind on subsites −2 to −1 and +2 to +3 in the E380Q/maltose complex, whereas they bind in tandem to −2 to −1 and +1 to +2 in the wild-type/maltose complex. The conformation of the glucose residue at subsite −1 was identified as a stable 4C 1 α-anomer in the E380Q/maltose complex, whereas a distorted ring conformation was observed in the wild-type/maltose complex. The side-chain movement of Gln380 to the position of a putative attacking water molecule seen in the wild-type enzyme caused the inactivation of the E380Q mutant and an altered binding pattern of maltose molecules. These results confirm the critical roles played by Glu186 in the donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in the activation of an attacking water molecule. The observed difference between the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342 suggests that the side-chain of Thr342 may stabilize the deprotonated form of Glu186 after the cleavage of the glycosidic bond.