Exposed Tryptophan Residues Research Articles

The effect of a variable initial pH on the structure and rheological properties of whey protein and monosaccharide gelation via the Maillard reaction

The effects of varying the initial pH from 5 to 9 on Maillard reaction-induced whey protein gel formation and structural and rheological properties were determined. The molecular mass of whey protein and reducing sugar conjugates increased according to SDS-PAGE determination. Amino and surface sulphydryl groups were decreased, contributing to gel network formation. The amide I and II bands at 1640 cm−1 and 1550 cm−1, respectively, were decreased and the amide III peak at 1200–1300 cm−1 increased, indicating the amino groups were participating in complex cascade reactions and the Maillard reaction during gel formation. Fluorescence measurements revealed that exposed tryptophan residues could increase the hydrophilic properties of the glycosylated protein. Gels formed from whey protein + glucose and from whey protein + xylose at an initial pH of 5 and 7, respectively, had a higher elastic modulus (G′), which was related to increased β-sheet and random coil formation after the Maillard reaction.

The exposed amino acids on protein skeleton control protein adsorption on surface-engineered silver nanoparticles

Adsorption of protein on the surface of silver nanoparticles (Ag NPs) could be controlled by modulating the surface using cyclodextrins (CDs). The CD nanocavities were tactically arranged over the Ag NPs with the help of surfactants. Two different model proteins, one with an exposed tryptophan residue and the other with the amino acid (tryptophan) captivated inside the folded structure, showed different motifs of surface adsorption based on host-guest interactions.

Label-Free Analysis of Protein Aggregation and Phase Behavior.

Phase transitions of protein molecules are central to biological function and malfunction. One such transition commonly encountered in nature is the conversion of soluble monomeric states into solid phases, which include crystals and amyloid fibrils, the latter of which are associated with the onset and development of neurodegenerative diseases. Monitoring aggregate formation and protein phase behavior is essential in gaining mechanistic insights into these fundamental processes. Fluorescence techniques have proven invaluable in observing biological molecules; yet, most such approaches rely on the use of an extrinsic fluorophore that binds to the molecule of interest, the installation of which can perturb the molecular systems under study. However, most proteins also possess aromatic amino acids within their peptide sequence and therefore exhibit intrinsic fluorescence. Here, we show that by measuring in space and time tryptophan autofluorescence for three proteins, reconstituted silk fibroin, β-lactoglobulin, and lysozyme, fibrillar self-assembly can be monitored accurately and without the need for extrinsic dyes. When fibrillar protein self-assembly takes place, hydrophobic burial occurs, resulting in the minimization of exposed tryptophan residues to the solvent and consequently leading to an increase in protein autofluorescence. Moreover, by employing a droplet-microfluidic approach to confine protein self-assembly in space, we demonstrate that intrinsic fluorescence can be used to image protein nanofibrils in a label-free manner and that the microstructural analysis obtained from intrinsic fluorescence microscopy correlates well with that from samples treated with extrinsic dyes. Finally, our results show that protein autofluorescence is not limited to the observation of β-sheet-rich structures, but can also be used to distinguish between different types of solid phases including spherulites and crystals, making this approach suitable for overall characterization of protein phase transition phenomena.

Complexation of lysozyme with lambda carrageenan: Complex characterization and protein stability

In a previous work, we reported that interaction of lysozyme (Lys) with kappa carrageenan (kCG) is governed by both electrostatic interactions and secondary forces, and lead to formation of asymmetric complex particles with an irregular localization of biopolymers without formation of a single center of binding. In this work we characterize complexation of lysozyme with strongly charged lambda carrageenan (lCG) and study protein stability within complex at different composition, pH and ionic strength (I) by a combination of a few main experimental techniques: turbidimetric titration, dynamic and electrophoretic light scattering, differential scanning microcalorimetry (DSMC), fluorescence and circular dichroism measurements. Complexation is governed by only electrostatic interactions, exhibits extremely low (7⋅10−4) qOnset values and lead to formation of swarm of large complex aggregates consisting of hundreds of very small separate particles. At pH 7 and I = 0.01 a stoichiometric lCG/Lys composition is equal to 0.309:1 (wt/wt) or 1:119 (mole/mole). It shift monotonically in the direction of higher lCG content with increase I values. The effect of I on stability of the complex formed has nonmonotonic character displaying a maximum at I ≈ 0.08. Complexation leads to a spectacular increase in the helix content and blue shift for Lys in the presence of lCG that probably reflects a more hydrophobic nonpolar environment of surface exposed tryptophan residues. It also significantly decreases thermal stability of Lys within complex in a wide range of pH suggesting that lCG has a higher affinity for the unfolded state than for the native state of Lys.

Protection of blue color in a spirulina derived phycocyanin extract from proteolytic and thermal degradation via complexation with beet-pectin

The focus of the presented work was on improvement of spirulina based phycocyanin (PhyC) extracts as a potential natural blue colorant by enhancement of color stability via polysaccharide complexation. Beet pectin (β-pectin), guar gum and soluble soy polysaccharides (SSPS) were assessed for their ability to protect the color of a commercial phycocyanin (PhyC) extract from thermal and proteolytic degradation. Both the beet pectin and guar gum studied formed stable and homogenous solutions with the PhyC extract; SSPS did not. Color change was assessed throughout by measurement of L, a, and b color space values (Hunter, 1948) before and after thermal or proteolytic treatment. Beet pectin improved color stability, over PhyC alone, with respect to heating at 65 °C (20 min) and proteolysis by Alcalase 2.4L, papain and bromelain. For these treatments beet pectin reduced color change values (ΔEab) from 13.0 ± 0.1 to 6.2 ± 0.4 following thermal treatment and from 12.7 ± 0.2 to 5.9 ± 0.7, 19.6 ± 1.3 to 12.5 ± 0.8, and 10.8 ± 0.4 to 4.6 ± 0.6 for the Alcalase, papain and bromelain treatments, respectively. Intrinsic fluorescence (excitation at 285 nm, emission at 325 nm) of beet pectin complexed PhyC indicates an increased presence of exposed tryptophan residues compared to PhyC alone. Furthermore, measured zeta-potentials following complex heating generally diverged to slightly more negative values for PhyC-beet pectin complexes at elevated temperatures compared to PhyC alone, suggesting that maintenance of a negatively charged environment may be important to pigment color stability in PhyC complexes.

Investigation of the binding interactions of Bisdemethoxycurcumin, Diacetylcurcumin and Diacetylbisdemethoxycurcumin with bovine α-lactalbumin by experimental and theoretical analysis

It was reported that bovine α-lactalbumin (BLA) as an important whey protein can be utilized as valuable vehicle for metal ions. The goal of this study was to investigate the interaction of BLA with bisdemethoxycurcumin (BDMC), Diacetylcurcumin (DAC), and diacetylbisdemethoxycurcumin (DABC) as three bioactive compounds by fluorescence quenching measurements and docking studies. It was observed that these ligands come closer to tryptophan residues and quench their emission without any change in their micro region polarity. The Stern–Volmer equation which is the best model to provide information about the interaction between small bioactive molecules and proteins was used to obtain the binding constants and the binding stoichiometry. Information about the extent of resonance energy transfer and Förster’s distance between donor and acceptor was estimated. Thermodynamic parameters confirmed that the final BDMC–BLA complex was stabilized by hydrogen bonds, whereas the final DABC–BLA and DAC–BLA complexes were stabilized by hydrophobic bonds which are in accordance with their chemical structures. Both the synchronous and docking studies verified that theTrp-26 which is the most exposed Tryptophan residue has the most contribution in the binding process. The Förster’s distances between bound ligands and tryptophans were in agreement with the measured distances by docking studies. The obtained achievements confirmed that there are considerable binding interactions between these curcuminoids and BLA.

Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics

Thermophilc mold Sporotrichum thermophile secretes an acidstable and thermostable phytase, which finds application as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. Low extracellular phytase production by the mold is a major bottleneck for its application on a commercial scale. We have successfully overcome this problem by constitutive secretary expression of codon optimized rStPhy under glyceraldehyde phosphate dehydrogenase (GAP) promoter in Pichia pastoris. A ∼41-fold improvement in rStPhy production has been achieved. Circular Dichroism (CD) spectra revealed that rStPhy is composed of 26.65% α-helices, 5.26% β-sheets and 68.09% random coils at pH 5.0 and 60°C, the optima for the enzyme activity. The melting temperature (Tm) of the enzyme is ∼73°C. The 3D structure of rStPhy displayed characteristic signature sequences (RHGXRXP and HD) of HAP phytase. The catalytically important amino acids (Arg74, His75, Arg78, His368 and Asp369) were identified by docking and site directed mutagenesis. Fluorescence quenching by N-bromosuccinimide (NBS) and CsCl exposed tryptophan residues surrounded by negative charges, which play a key role in maintaining structural integrity of rStPhy.

Use of Spectroscopic Techniques to Reveal the Nature of the Interactions of Two Sialic Acid Specific Lectins with Gold Nanoparticles.

From UV-vis absorption, steady state and time resolved fluorescence measurements coupled with circular dichroism (CD) spectral studies, it was revealed that among the two lectins: Sambucus nigra agglutinin (SNA) and Saraca indica (saracin II), SNA forms stronger binding complex in the ground state with gold nanoparticles (GNPs). From the measurements of Stern-Volmer (SV) constants Ksv, and binding constants K(A) and number of binding sites two important inferences could be drawn. Firstly, the fluorescence quenching is primarily due to static quenching and secondly SNA forms stronger binding with GNPs relative to the other lectin saracin II. Synchronous fluorescence spectral measurements further substantiate this proposition of exhibiting the fully exposed tryptophan residue in case of SNA. It appears that the lectin SNA adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with GNPs. CD measurements demonstrate that gold nanoparticles when interact with the lectins (glycoproteins), no significant distortion in the structural pattern of the later occurs. The unaltered identity in the secondary structural pattern of both SNA and saracin II in presence of gold nanoparticles hints that GNPs may be used as useful drug or drug delivery systems.

Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in species of the order Polyporales.

Structural analysis of a putative family 32 carbohydrate-binding module from the Streptococcus pneumoniae enzyme EndoD.

EndoD is an architecturally complex endo-β-1,4-N-acetylglucosamidase from Streptococcus pneumoniae that cleaves the chitobiose core of N-linked glycans and contributes to pneumococcal virulence. Although the glycoside hydrolase family 85 catalytic module has been structurally and functionally characterized, nothing is known about the ancillary modules and how they contribute to the overall function of the enzyme. Presented here is the 2.0 Å resolution structure of a family 32 carbohydrate-binding module of EndoD, SpCBM32, solved by single-wavelength anomalous dispersion. The putative binding site of this protein is a charge-neutral relatively flat region on the protein surface that contains one prominently exposed tryptophan residue that extends into the solvent. These topographical features are discussed in the biological context of EndoD activity and a hypothesis is made about the complex structure of its potential carbohydrate ligand.

Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

The intracellular enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Pseudomonas aeruginosa is a potential drug target because it is essential for the growth of bacteria while it is absent in humans. Therefore, in order to design new compounds using structure based approach for inhibiting the function of DHDPS from P. aeruginosa ( Ps), we have cloned, characterized biochemically and biophysically and have determined its three-dimensional structure. The gene encoding DHDPS ( dapA) was cloned in a vector pET-28c(+) and the recombinant protein was overexpressed in the Escherichia coli host. The K m values of the recombinant enzyme estimated for the substrates, pyruvate and ( S)-aspartate-β-semialdehyde [( S)-ASA] were found to be 0.90 ± 0.13 mM and 0.17 ± 0.02 mM, respectively. The circular dichroism studies showed that the enzyme adopts a characteristic β/α conformation which is retained up to 65 °C. The fluorescence data indicated the presence of exposed tryptophan residues in the enzyme. The three-dimensional structure determination showed that DHDPS forms a homodimer which is stabilized by several hydrogen bonds and van der Waals forces at the interface. The active site formed with residues Thr44, Tyr107 and Tyr133 is found to be stereochemically suitable for catalytic function. It may be noted that Tyr107 of the catalytic triad belongs to the partner molecule in the dimer. The structure of the complex of PsDHDPS with ( S)-lysine determined at 2.65 Å resolution revealed the positions of three lysine molecules bound to the protein.

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.

Familial lipoprotein lipase deficiency caused by known (G188E) and novel (W394X) LPL gene mutations

Lipoprotein lipase (LPL) is the key enzyme in the catabolism of triglyceride-rich lipoproteins in the circulation. Familial LPL deficiency is characterized by hypertriglyceridaemia and absence of LPL activity. We report a case of LPL deficiency in a 43-year-old woman, who initially presented in childhood with chylomicronaemia syndrome. At that time, her plasma triglyceride concentration was approximately 30 mmol/L and post-heparin lipolytic activity was very low. In addition to having the known missense mutation LPL G188E, the patient was also found to have a novel nonsense mutation in exon 8, namely LPL W394X. The novel substitution in exon 8 (c.1262G > A) predicts a truncated protein product of 393 amino acids that lacks the carboxylterminal 12% of the mature LPL. Trp(394) is part of a cluster of exposed tryptophan residues in the carboxyl-terminal domain of LPL important for binding lipid substrate. Of 11 members from her three-generation family, three were heterozygotes for G188E (mean plasma triglyceride, 3.5 +/- 2.0 mmol/L), whereas six were heterozygotes for W394X (triglyceride, 4.3 +/- 1.8 mmol/L). In summary, we describe a case of familial LPL deficiency caused by compound heterozygosity for known (G188E) and novel (W394X) LPL gene mutations.

Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.

Contribution of N-Linked Glycans to the Conformation and Function of Intercellular Adhesion Molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.

Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus

The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K(m) of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.

Stabilization and characterization of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae

Histidine-tagged homocitrate synthase from Saccharomyces cerevisiae was purified to about 98% using a Ni–NTA resin and stabilized using a combination of 100 mM guanidine hydrochloride, 100 mM α-cyclodextrin, and 600 mM ammonium sulfate. The enzyme was assayed using dichlorophenol indophenol (DCPIP) as an oxidant to oxidize the CoASH produced in the reaction. A stoichiometry of 1:1 was obtained between DCPIP and CoASH. Kinetic parameters for the stable enzyme at pH 7.5 are: K m (AcCoA), 24 μM; K m (α-kg), 1.3 mM; and k cat, 37 min −1. The enzyme, in the absence of reactants, self-associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed tryptophan residue and a mixed (α/β) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide suggest that the microenvironment around the single tryptophan residue of the enzyme has some positive charge.

Human glycine N-methyltransferase is unfolded by urea through a compact monomer state

Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0–3.5 M urea with enzyme inactivation. The compact monomers were characterized by Stokes radius ( R s) of 40.7 Å equal to that of globular proteins with the same molecular mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R s of 50 Å, exposed tryptophan residues, and disrupted secondary structure in 8 M urea.

Structural Analysis of Lipid Complexes of GM2-Activator Protein

The GM2-activator protein (GM2-AP) is a small lysosomal lipid transfer protein essential for the hydrolytic conversion of ganglioside GM2 to GM3 by β-hexosaminidase A. The crystal structure of human apo-GM2-AP is known to consist of a novel β-cup fold with a spacious hydrophobic interior. Here, we present two new structures of GM2-AP with bound lipids, showing two different lipid-binding modes within the apolar pocket. The 1.9Å structure with GM2 bound shows the position of the ceramide tail and significant conformational differences among the three molecular copies in the asymmetric unit. The tetrasaccharide head group is not visible and is presumed to be disordered. However, its general position could be established through modeling. The structure of a low-pH crystal, determined at 2.5Å resolution, has a significantly enlarged hydrophobic channel that merges with the apolar pocket. Electron density inside the pocket and channel suggests the presence of a trapped phospholipid molecule. Structure alignments among the four crystallographically unique monomers provide information on the potential role for lipid binding of flexible chain segments at the rim of the cavity opening. Two discrete orientations of the S130-T133 loop define an open and a closed configuration of the hydrophobic channel that merges with the apolar pocket. We propose: (i) that the low-pH structure represents an active membrane-binding conformation; (ii) that the mobile S130-T133 loop serves as a gate for passage of ligand into the apolar pocket; and (iii) that this loop and the adjacent apolar V59-W63 loop form a surface patch with two exposed tryptophan residues that could interface with lipid bilayers.

Role of a solvent-exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo-beta-lactamase.

Numerous X-ray crystal structures of the metallo-beta-lactamase from Bacteroides fragilis and related organisms show a beta-hairpin loop immediately adjacent to the active-site zinc atom(s). Both crystallographic and NMR information show that the end of this beta-hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X-ray structures to a lack of observable electron density in this region. We report an investigation of the role of this mobile, solvent-exposed tryptophan using site-directed mutagenesis, steady state kinetics measurements and characterization by NMR. Trp49 appears to have a role both in substrate binding and in promotion of catalysis. Substitution of this residue with a number of different side chains indicates that the binding interaction depends on the bulky hydrophobic and aromatic nature of the indole ring, which can provide relatively non-specific interactions with a variety of antibiotic substrates. In this way, the tryptophan at this position provides a large degree of the breadth of substrate specificity for the metallo-beta-lactamase. Previous studies established that the antibiotic binding site was sufficiently plastic that the derivatization of existing antibiotics is unlikely to result in the successful treatment of bacterial infections incorporating this resistance element. Rather, a more productive approach may be to design therapeutics directed towards this solvent-exposed tryptophan residue.