Michael David Lee

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein we report the dual site-specific labelling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labelling.

Spin labels containing a Gd(III) ion have become important for measuring nanometer distances in proteins by double electron–electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(III) tag used. Here we report the performance of two Gd(III) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B–NS3 protease than previously established Gd(III) tags. The improved performance is consistent with the absence of specific hydrophobic or charge–charge interactions with the protein. In the case of the Zika virus NS2B–NS3 protease, unexpectedly broad Gd(III)–Gd(III) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B–NS3 protease, both in the presence and absence of an inhibitor.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8‐dihydropteroate (DHPt) from 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphate (DHPPP) and para‐aminobenzoic acid (pABA). DHPS is the long‐standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild‐type and sulfa drug resistant strains. Following the work on developing pterin‐site inhibitors of the adjacent enzyme 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8‐mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub‐μm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand‐bound EcDHPS crystal structures delineate the structure–activity relationship observed providing a structural framework for the rational development of novel, substrate‐envelope‐compliant DHPS inhibitors.

A new pair of enantiomeric two-armed lanthanide-binding tags have been developed for paramagnetic NMR studies of proteins.

Double‐arm cyclen‐based Gd³⁺ tags are shown to produce accurate nanometer scale Gd³⁺–Gd³⁺ distance measurements in double electron–electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α‐helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd³⁺–Gd³⁺ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double‐arm Gd³⁺ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective S_P and R_P diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the S_P than the R_P oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

Methods based on pulse electron paramagnetic resonance allow measurement of the electron–electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron–Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(III)–Gd(III) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(III) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(III) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(III) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.

A new pair of two-armed lanthanide-binding tags provide distinct sets of structural restraints when attached to the same site of a protein.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 μM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

Double electron–electron resonance (DEER) is used to probe structure of Gd³⁺-tagged biomolecules by determining Gd³⁺–Gd³⁺ distances.