Edited by Norma AllewellThe Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs inFnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/k off ) and dissociation constants (K d ‫؍‬ k off /k on ), determined by mechanically dissociating the Fn⅐peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 M) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 M). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (K d app ‫؍‬ 0.2-0.5 M, as determined by SPR) compared with the lowest-affinity double-alanine peptide (K d app ‫؍‬ 3.8 M). Together, these findings demon-. 4 The abbreviations used are: Fn, fibronectin; AFM, atomic force microscopy; COM, center of mass; CV, collective variable; FnBPA, fibronectin-binding protein A; FnBR-9, fibronectin-binding repeat 9 in FnBPA; MD, molecular dynamics; MSCRAMM, molecular surface component recognizing adhesive matrix molecule; NTD, N-terminal domain; SPR, surface plasmon resonance; nN and pN, nanonewton and piconewton, respectively. cros ARTICLE

(18-Crown-6)-2,3,11,12-tetracarboxylic acid is a useful chiral NMR solvating agent for isoxazoline-fused β-amino acid derivatives. Isoxazoline substrates are analyzed as their hydrochloride salts in methanol-d(4). The crown ether and substrate associate through the formation of three hydrogen bonds between the protonated amine and crown ether oxygen atoms. Enantiomeric discrimination is observed for two or more resonances of every substrate. At least one of these resonances is free of overlap with other resonances in the spectrum and has large enough enantiomeric discrimination to enable the determination of enantiomeric purity. 2D COSY methods can be used to identify additional resonances that exhibit enantiomeric discrimination in the NMR spectrum.