Christopher M. Clouthier

Peptide self-assembled monolayers for label-free and unamplified surface plasmon resonance biosensing in crude cell lysate.

Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/mL, respectively. Minimizing these nonspecific interactions has allowed rapid and direct quantification of beta-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequently form a monolayer on gold in the form of 3-MPA-(AA)(n)-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-(AA)(n)-OH showed that the lowest nonspecific adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-(Ser)(5)-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from beta-lactamase by approximately 1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar beta-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media.

Identification and Characterization of an Inborn Error of Metabolism Caused by Dihydrofolate Reductase Deficiency

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.

Coumarin-Based Fluorogenic Probes for No-Wash Protein Labeling†

A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.

Acyl transfer mechanisms of tissue transglutaminase.

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that catalyses several acyl transfer reactions. The most biologically relevant of these involve protein-bound Gln residues as an acyl-donor substrate, and either water or a primary amine as an acyl-acceptor substrate. The former leads to deamidation of Gln to Glu, whereas the latter leads to transamidation, typically resulting in protein cross-linking when the amine substrate is a protein-bound Lys residue. In this review, we present an overview of over fifty years of mechanistic studies that have led to our current understanding of TG2-mediated hydrolysis and transamidation.

Real‐Time Monitoring of Protein Conformational Dynamics in Solution Using Kinetic Capillary Electrophoresis

Conformational analysis: Capillary electrophoresis (CE) allows for the rapid separation of slowly interconverting protein conformers. Kinetic analysis (k(open), k(closed), and K(d)) of electropherograms in the presence and absence of effector ligands allows the measurement of kinetic and thermodynamic constants associated with conformational changes and ligand binding.

Maintenance of Native-like Protein Dynamics May Not Be Required for Engineering Functional Proteins.

Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

Expression, purification and kinetic characterisation of human tissue transglutaminase.

The expression of soluble recombinant transglutaminase (TGase) has proven to be a challenge for many research groups. Herein, we report a complementary method for the expression, in BL21(DE3) Escherichia coli, of recombinant human tissue transglutaminase (hTG2) whose solubility is enhanced through N-terminal fusion to glutathione S-transferase (GST). Moreover, we report the cleavage of the GST tag using PreScission™ Protease (PSP) and purification of hTG2 in its untagged form, distinctively suitable for subsequent studies of its remarkable conformational equilibrium. The effects of co-solvents and storage conditions on stability of purified hTG2 are also reported. Furthermore, we demonstrate for the first time the use of a convenient chromogenic assay to measure the activity of the human enzyme. The utility of this assay was demonstrated in the measurement of the kinetic parameters of a wide variety of substrates and inhibitors of both hTG2 and the extensively studied guinea pig liver TGase. Finally, comparison of these results provides further evidence for the functional similarity of the two enzymes.

Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE-UV-IM-MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE-UV-IM-MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry†

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA–metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes. KCE-MS separates a thrombin-binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveals the apparent equilibrium folding constant (KF≈150 μm), folding rate constant (kon≈1.70×103 s−1 m−1), unfolding rate constant (koff≈0.25 s−1), half-life time of the G-quadruplex (t1/2≈2.8 s), and relaxation time (τ≈3.9 ms at physiological 150 mm [K+]). In addition, KCE-MS screens for a GQ-stabilizing/-destabilizing effect of DNA binding dyes and an anticancer drug, cisplatin.