A near-infrared excitation/emission fluorescent probe for imaging of endogenous cysteine in living cells and zebrafish.

The fluorescence imaging technique provides an essential tool for studying biological systems. However, due to the interference of autofluorescence of biological tissues, the application of short-wavelength fluorescent probes in biological imaging was limited. The near-infrared (NIR) excitation/emission fluorescent probe possesses unique advantages in optical imaging in vivo, including less light scattering, minimal photo-damage to biological samples, deep tissue penetration, and weak autofluorescence interference from complicated biological systems. In this work, a convenient fluorophore (E)-2-[2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3- methylbenzo[d]thiazol-3-ium iodide (DXM-OH) with NIR excitation and emission was rationally designed and developed. What's more, DXM-OH was applied to construct an "OFF-ON" fluorescent probe (E)-2-{2-[6-(acryloyloxy)-2,3-dihydro-1H- xanthen-4-yl]vinyl}-3-methylbenzo[d]thiazol-3-ium iodide (DXM) for sensitive and selective detection of cysteine (Cys). The experimental results showed that DXM had the advantages of good cell permeability, low toxicity, and excellent optical properties (NIR excitation/emission) and it was successfully applied to image Cys of living cells and zebrafish.

GPMVs as a Tool to Study Caveolin-Interacting Partners.

Caveolins, major components of small plasma membrane invaginations called caveolae, play a role in signaling, particularly in mechanosignaling. These proteins are known to interact with a variety of effector molecules, including G-protein-coupled receptors, Src family kinases, ion channels, endothelial nitric oxide synthase (eNOS), adenylyl cyclases, protein kinase A (PKA), and mitogen-activated PKs (MAPKs). There is, however, speculation on the relevance of these interactions and the mechanisms by which caveolins may control intracellular signaling. This chapter introduces a method of isolation of giant plasma membrane-derived vesicles (GPMVs), which possess full complexity of membrane they originate from, thus comprising an excellent platform to revisit some of the previously described interactions in a cleaner environment and possibly identifying new binding partners. It is also a powerful technique for studying membrane mechanics, as it was previously used to demonstrate the role of caveolae in mechanoprotection.

Ag+ Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.

Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the ß-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the ß-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the ß-domain, but our data suggest the two additional Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7-17) are formed in solutions that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addition of NEM to this solution to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag+ increases.

Comparison of N-ethyl maleimide and N-(1-phenylethyl) maleimide for derivatization of biological thiols using liquid chromatography-mass spectrometry.

The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents: N-ethyl maleimide (NEM) and (R)-(+)-N-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine, N-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized. Graphical abstract.

Radiolabelling of lipid-based nanocarriers with fluorine-18 for in vivo tracking by PET.

Organic nanoparticles made out of biodegradable and biocompatible materials have attracted increased attention in the therapeutic and diagnostic fields. In this study, we attempted to explore a new radiolabelling chelating free strategy for biodegradable sphingomyelin nanometric emulsions with fluorine-18 (18F), a radioisotope regularly used in clinic. [18F]fluoride was produced by the cyclotron and was incorporated into 4-[18F]fluorobenzamido-N-ethylmaleimide ([18F]FBEM), which was coupled next to the emulsions previously functionalized with a thiol group, via inclusion of either a thiol-PEG-lipid (SH-PEG12-C18), or a peptide-PEG-lipid (Cys-Pro-Ile-Glu-Asp-Arg-Pro-Met-Cys-PEG8-C18) derivative. Radiolabelled emulsions were obtained in a rapid and efficient fashion through facile-conjugated chemistry without the use of organic solvents, and characterized in terms of size, polydispersity, surface charge, pH, and osmolarity. PET imaging and biodistribution studies in BALB/c mice allowed obtaining the pharmacokinetics of the radiolabelled emulsions and determining the clearance pathways. Altogether, we confirmed the potential of this new technique for the radiolabelling of lipid-based drug nanosystems for application in PET imaging diagnosis.

Highly specific monitoring and imaging of endogenous and exogenous cysteine in living cells.

Cys is one of the important biothiols and its abnormal concentration may pose a threat to human health. Therefore, the monitoring of Cys in organisms is of great significance. GSH and Hcy, as the other two biothiols, have similar chemical structures and active sites to Cys. Consequently, developing fluorescent probes to independently detect Cys has become a challenging problem. Keeping this in mind, α-ß unsaturated ketone as a recognition group was integrated into the coumarin group skeleton to synthesize a fluorescent probe SC. After the nucleophilic addition reaction of Cys with SC, the conjugated system of SC was blocked and the fluorescent enhanced obviously. SC was able to detect Cys specifically under the same excitation with a low detection limit (11.1 nM). SC showed a rapid respond to Cys (120 s) and good fluorescent stability over a wide pH range. In addition, it achieved extracorporeal circulation in the presence of H2O2 or NEM. In the end, SC could be applied to detecting endogenous and exogenous Cys under biological condition due to its slight cytotoxicity and good biocompatibility. This provided a powerful tool for studying the physiological function of Cys exclusively.

Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols.

The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Km = 0.31) and ficin-TN (Km = 0.39) to H2O2 compared with ficin (Km = 0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01-16 µM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.

Stability of nitroxide biradical TOTAPOL in biological samples.

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

A redox sensitivity-based method to quantify both pentameric and monomeric C-reactive protein in a single assay.

C-reactive protein (CRP) can exist in both pentameric (pCRP) and monomeric conformation (mCRP). Though serum pCRP is an established marker of inflammation, the diagnostic significance of mCRP remains unknown largely due to the lack of a reliable assay. The power and specificity of antibody-based assays are limited by the antibody reagents used and by the degree of cross-reactivity that may exist in detecting each antigen, as mCRP is known to be formed from the pentameric and both conformations usually coexist in clinical samples. Here, we describe an assay that measures both CRP conformations in simple samples in a single assay. This assay depends on the rationale that the intra-molecular disulfide bonds in pCRP resist reduction, while those in mCRP can be readily reduced. The distinct sensitivity of pCRP and mCRP to reduction can be easily detected and separated by electrophoresis. This assay may provide a means to study clinical correlation between pCRP and mCRP in clinical samples in the future and to evaluate their respective significance as disease markers.

Sulfhydryl Labeling as a Tool to Investigate the Topology of Membrane Proteins Involved in Lipopolysaccharide Biosynthesis.

Establishing the topology of membrane proteins, especially when their tridimensional structures are unavailable, is critical to identify functional regions, delimit the protein orientation in the membrane, the number of transmembrane segments, and the position of critical amino acids (whether exposed to the solvent or embedded in the lipid bilayer). Elucidating the topology of bacterial integral membrane proteins typically involves the construction of deletion-fusions whereby regions of the protein are fused to reporters. Although these methods have several advantages, they are also artifact prone. In contrast, methods based on single amino acid substitutions preserve the native protein intact. We describe here an assay to analyze the topology of membrane proteins involved in the biogenesis of bacterial glycoconjugates, which is based on the accessibility of cysteine substitutions at various places in the protein under in vivo and in vitro conditions. Cysteine residues are detected with polyethylene glycol-maleimide (PEG-Mal). This procedure can be applied to crude bacterial cell extracts and does not require protein purification.

Biophysical phenotyping of single cells using a differential multiconstriction microfluidic device with self-aligned 3D electrodes.

Precise measurement of mechanical and electrical properties of single cells can yield useful information on the physiological and pathological state of cells. In this work, we develop a differential multiconstriction microfluidic device with self-aligned 3D electrodes to simultaneously characterize the deformability, electrical impedance and relaxation index of single cells at a high throughput manner (>430 cell/min). Cells are pressure-driven to flow through a series of sequential microfluidic constrictions, during which deformability, electrical impedance and relaxation index of single cells are extracted simultaneously from impedance spectroscopy measurements. Mechanical and electrical phenotyping of untreated, Cytochalasin B treated and N-Ethylmaleimide treated MCF-7 breast cancer cells demonstrate the ability of our system to distinguish different cell populations purely based on these biophysical properties. In addition, we quantify the classification of different cell types using a back propagation neural network. The trained neural network yields the classification accuracy of 87.8% (electrical impedance), 70.1% (deformability), 42.7% (relaxation index) and 93.3% (combination of electrical impedance, deformability and relaxation index) with high sensitivity (93.3%) and specificity (93.3%) for the test group. Furthermore, we have demonstrated the cell classification of a cell mixture using the presented biophysical phenotyping technique with the trained neural network, which is in quantitative agreement with the flow cytometric analysis using fluorescent labels. The developed concurrent electrical and mechanical phenotyping provide great potential for high-throughput and label-free single cell analysis.

Selective Extraction and Antioxidant Properties of Thiol-Containing Peptides in Soy Glycinine Hydrolysates.

A highly selective procedure to extract thiol-containing peptides (TCPs) from complicated soy glycinin hydrolysates (SGHs) was described. This procedure included the reduction of disulfide bonds by 1,4-dithiothreitol (DTT) and enrichment of TCPs through Thiopropyl-Sephrose 6B covalent chromatography. TCPs were confirmed using a strategy based on mass shift after differential alkylation of sulfhydryl groups with iodoacetamide and N-ethylmaleimide by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The antioxidant activities of TCPs were evaluated using chemical assays. DTT reduction increased the concentration of sulfhydryl groups from 1.8 µmol/g to 113.8 µmol/g. The efficiency of the extraction was improved by optimizing the loading of sample, extraction and desorption time and the content of desorption reagent. Both of the adsorption and desorption process were found to fit a pseudo-second order model. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs. The EC50 of TCPs for DPPH, hydroxyl radical, and superoxide anion radical was 0.1, 1.49 and 0.084 mg/mL, respectively. The reducing power of TCPs (0.2 mg/mL) was of 0.375. These results suggest that the combination of DTT reduction and Thiopropyl-Sepharose 6B covalent chromatograph was a successful pathway to extract TCPs from SGHs and the TCPs could be used as potential antioxidants.

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Chromatographic separation of similar post-translationally modified metallothioneins reveals the changing conformations of apo-MT upon cysteine alkylation by high resolution LC-ESI-MS.

Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated ß-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the ß-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release.

Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44.

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

Proteomic profiling of oxidized cysteine and methionine residues by hydroxyl radicals in myosin of pork.

In order to investigate the effects of hydroxyl radicals on the myosin of pork, with focus on reducible and non-reducible oxidation of specific cysteine and methionine residues, extracted myofibrillar protein from longissimus dorsi of pork was incubated with H2O2 for 24, 48 and 72h, respectively. The thiol contents and crosslinking of myofibrillar protein were analyzed after oxidation of the protein. Moreover, cysteine (labeled with N-ethylmaleimide (NEM) and iodoacetamide (IAM) before and after reduction, respectively) and methionine oxidation were detected by LC/MS using label-free quantitation. The result revealed that cysteine at head of myosin tended to form sulfinic and sulfonic acid, while the cysteine at coiled tail of myosin easily generated disulfide under same condition. Furthermore, it was also revealed that the methionine at the coiled tail of myosin was more easily oxidized than that of the head.

Analysis of Disulfide Bond Formation.

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, thiopropyl-sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations.

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein's conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.