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)

Abstract

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.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…The replacement of tryptophan by HQA at position 10 in the sequence of full-length CXCR1 (1-350) was performed as previously described for a different construct of CXCR1 (56). The complete replacement of tryptophan by HQA was confirmed by NMR and fluorescence spectroscopies.…”

“…The probe temperature was maintained at 8 C using nitrogen cooling gas at À30 C and a flow rate of 1500 L/h; the actual sample temperature, as monitored by the resonance frequency of the water signal, was estimated to be 30 C due to frictional heating. Two-dimensional 1 H-detected 1 H-15 N chemical shift correlation spectra were acquired using either a 1 H-15 N heteronuclear single quantum correlation (HSQC) pulse sequence via double insensitive nuclei enhanced by polarization transfers (INEPT) (59) with presaturation for water signal suppression, or via double cross-polarization (CP) transfers (46,56) with multiple intense solvent suppression intended for sensitive spectroscopic investigation of protonated proteins instantly (47). 15 N globally optimized alternating phase rectangular pulse (60) decoupling with irradiation of 22.6 kHz was applied during 1 H signal acquisition.…”

“…The genetic incorporation of the metal-chelating unnatural amino acid HQA (67) into membrane proteins enables the measurement of intra-and intermolecular distance restraints by enabling PRE NMR experiments (56). The signal intensities in the one-dimensional MAS solid-state 15 N-edited 1 H NMR spectrum of IL-8 (1-66) bound to W10HQA CXCR1 (1-350) (Fig.…”

“…Changes observed in spectra of IL-8 (1-66) upon binding CXCR1 (1-350) and NT-CXCR1 (39-350) identify chemokine residues involved in the interactions with CXCR1. In addition, we incorporated the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid dihydrochloride (HQA) into full-length CXCR1, expanding upon earlier work (56), to observe intermolecular paramagnetic relaxation enhancement (PRE) between IL-8 (1-66) and CXCR1 . This provides additional information about the structures and spatial arrangement of the complexed proteins.…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…Furthermore, the site-specific incorporation of the metal-binding ncAA, 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid ( Fig. 1B21), into membrane proteins (CXCR1 and viroporin p7) allowed long-range paramagnetic relaxation measurements of intra-and intermolecular distance (Park et al 2014). In related work, to facilitate electron paramagnetic resonance (EPR) studies of proteins, a ketoxime-linked spin label side chain has been site-specifically conjugated to proteins through a reactive chemical handle (Fleissner et al 2009).…”