A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

Abstract

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

“…Similar approaches were used on a LecA protein interacting with galactose linked to TOTAPOL, and dihydrofolate reductase (DHFR) interacting with its ligand trimethoprim (TMP) covalently linked with TOTAPOL . However, in these cases, significant NMR signal quenching may be encountered in the vicinity of the binding site. ,,,, Very recently, a method for insertion of a nitroxide spin label into disulfide bridges in proteins has been introduced . For membrane proteins, an approach where PAs are covalently bound to a lipid can be used; the reconstitution with such lipids allows a homogeneous incorporation of the PA into a membrane bilayer. , …”

“…98,156,158,329,330 Very recently, a method for insertion of a nitroxide spin label into disulfide bridges in proteins has been introduced. 331 For membrane proteins, an approach where PAs are covalently bound to a lipid can be used; the reconstitution with such lipids allows a homogeneous incorporation of the PA into a membrane bilayer. 332,333 Sani et al have utilized solid-phase synthesis to prepare a doubly spin-labeled variant of the antimicrobial peptide maculatin 1.1 (Mac1).…”

Biomolecular and Biological Applications of Solid-State NMR with Dynamic Nuclear Polarization Enhancement

“…DNP enhancement was demonstrated on the glycerol signals of the DNP matrix. Because eptifibatide was naturally abundant, and potentially subject to bleaching from the radical, it was not observed directly under the conditions of the experiment …”

“…Because eptifibatide was naturally abundant, and potentially subject to bleaching from the radical, it was not observed directly under the conditions of the experiment. 156 As the indirect polarization pathway relies on spin diffusion, the degree of selectivity that targeted DNP can achieve is limited. In addition, the signal intensity of nuclear spins in the closest vicinity to the radical tag are strongly affected by bleaching effect due to paramagnetic relaxation enhancement (PRE) and may not be detectable in targeted approaches.…”

A disintegrin derivative as a case study for PHIP labeling of disulfide bridged biomolecules

“…Herein, we demonstrate by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI–MS) that the intercalator 2 can be incorporated into the disulfide bridge of the peptide. Recently it has been shown that eptifibatide can be efficiently spin-labeled for hyperpolarization applications employing Dynamic Nuclear Polarization 50 . In the present paper we demonstrate that it is also feasible to employ derivatives of eptifibatide as efficient hyperpolarization sources in PHIP-NMR spectroscopy.…”