A Two‐Armed Lanthanoid‐Chelating Paramagnetic NMR Probe Linked to Proteins via Thioether Linkages

Liu, Weimin; Skinner, Simon P.; Timmer, Monika; Blok, Anneloes; Hass, Mathias A. S.; Filippov, Dmitri V.; Overhand, Mark; Ubbink, Marcellus

doi:10.1002/chem.201400257

Cited by 32 publications

(40 citation statements)

References 24 publications

(38 reference statements)

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“…In the structure of the closed state, the lanthanoid is positioned ∼8 Å from the Cα atoms of the two Cys residues (Fig. S3A), consistent with the results of other studies using CLaNP tags (34,(36)(37)(38). In contrast, in the fit of the PCS data to the structure of the open state, the paramagnetic center is placed far from the expected position, due to the moved G-helix (Fig.…”

supporting

confidence: 89%

Delicate conformational balance of the redox enzyme cytochrome P450cam

Skinner

Liu

Hiruma

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.uring catalysis, an enzyme will traverse a conformational energy landscape that can be highly complex and easily changed by external factors, such as temperature, ionic strength, and crystallization. Cytochromes P450 (CYPs) are b-type hemecontaining monooxygenases found throughout the three domains of life. These enzymes catalyze the regiospecific and stereospecific hydroxylation of various aliphatic and aromatic compounds and are involved in a considerable number of metabolic processes, such as steroid biosynthesis and metabolism of xenobiotics in mammals. The CYP superfamily has been extensively studied over the past five decades, and an understanding of its mechanism is crucial for the development of pharmaceutical compounds that do not inhibit these enzymes. Under other circumstances, it is desirable to inhibit a CYP for therapeutic reasons. For example, compounds that inhibit CYP1B1 and CYP1A2 in humans have been proposed as promising anticancer agents (1). The archetypal member of this superfamily is CYP101A1 (more commonly known as P450cam) from Pseudomonas putida, which was the first CYP for which the 3D structure was determined by X-ray crystallography (2). P450cam catalyzes the hydroxylation of D-camphor to 5-exohydroxycamphor using two electrons, two protons, and molecular oxygen. Putidaredoxin (Pdx) reductase oxidizes NADH and transfers the electrons to Pdx, which, in turn, shuttles electrons to P450cam in two consecutive steps (3-5). This complex reaction is controlled by the enzyme to ensure an efficient coupling between dioxygen reduction and substrate hydroxylation and to avoid side reactions. P450cam must go through a cycle of several conformational and electronic states to perform its catalytic task (6).The first X-ray crystal structures of P450cam showed that the enzyme occupied a closed conformation, both in the presence and absence of substrate; therefore, it was unclear how the substrate was able to bind into the active site (2). Moreover, structures of the intermediates of the catalytic cycle were also in a closed conformation (7), leading to the idea that during the catalytic cycle, P450cam opens to bind camphor, closes to perform hydroxylation, and then re...

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supporting

confidence: 89%

Delicate conformational balance of the redox enzyme cytochrome P450cam

Skinner

Liu

Hiruma

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…CLaNP-9 tag is unstable during lyophilization and that the tag was immediately used for protein labeling following purification. 9 In our hands, lyophilization of purified NMR Experiments. All NMR spectra were recorded at 10 o C on a Bruker 800 MHz NMR spectrometer equipped with a cryoprobe using 50 µM solutions of DNA duplex in 3 mm NMR tubes.…”

Section: Tag Stability Liu Et Al Reported That Their α-Bromoacetamimentioning

confidence: 97%

“…3,4,6 Alternatively, electrophilic tags have been designed that form a thioether linkage, which is chemically more stable and also produces a shorter linker between the sulfur atom and the lanthanide ion. [7][8][9][10][11] Attaching lanthanide-binding tags to DNA or RNA is more difficult because native nucleotides contain no thiol groups. Obviously, this limitation does not hold for synthetic DNA fragments, which can be furnished with a wide variety of non-natural functional groups.…”

Section: Introductionmentioning

confidence: 99%

“…Initial attempts to tag the PT oligonucleotides with compounds containing activated disulfide bonds (such as the C1 tag 22 ) failed. As a previous report showed successful alkylation of PT oligonucleotides with iodoacetamide-PROXYL, 18 we produced the C10 tag ( Figure 3), which is a cyclen-based tag featuring an alkylating group akin to that found in the recently reported CLaNP-9 tag of Liu et al 9 Like earlier cyclen tags, [24][25][26] it contains chiral phenylethylacetamide groups to populate a single stereoisomer of the lanthanide complex. Ligation reactions using a five-fold excess of tag (loaded with either Tb 3+ , Tm 3+ or Y 3+ ) produced tagged DNA in practically quantitative yield in an overnight reaction ( Figure S5).…”

Section: Introductionmentioning

confidence: 99%

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New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group

Lee

Carruthers

et al. 2017

Bioconjugate Chem.

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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 and R 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 than the R 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.

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“…However, since many tags are more flexible than a typical chemical coordination site, it is essential to restrict their dynamics. This has been achieved with bulky tags, including peptide tags as discussed above, two-point attached tags (Liu et al 2014b), and tags that interact with protein side chains (Su et al 2008). With dynamically restricted lanthanide tags, significant PCSs can be observed over very large distances, perhaps beyond 100 Å (Hass and Ubbink 2014).…”

Section: Paramagnetic Protein Nmrmentioning

confidence: 99%

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

et al. 2014

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The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid (UAA) that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl) propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

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A Two‐Armed Lanthanoid‐Chelating Paramagnetic NMR Probe Linked to Proteins via Thioether Linkages

Abstract: Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.

Cited by 32 publications

References 24 publications

Delicate conformational balance of the redox enzyme cytochrome P450cam

Delicate conformational balance of the redox enzyme cytochrome P450cam

New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

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