Slow Aromatic Ring Flips Detected Despite Near-Degenerate NMR Frequencies of the Exchanging Nuclei

Abstract

Aromatic ring flips of Phe and Tyr residues are a hallmark of protein dynamics with a long history in molecular biophysics. Ring flips lead to symmetric exchange of nuclei between sites with distinct magnetic environments, which can be probed by NMR spectroscopy. Current knowledge of ring-flip rates originates from rare cases in which the chemical shift difference between the two sites is sufficiently large and the ring-flip rate sufficiently slow, typically kflip < 10(3) s(-1), so that separate peaks are observed in the NMR spectrum for the two nuclei, enabling direct measurement of the flip rate. By contrast, a great majority of aromatic rings show single peaks for each of the pairs of δ or ε nuclei, which commonly are taken as inferential evidence that the flip rate is fast, kflip < 10(3) s(-1), even though rate measurements have not been achieved. Here we report a novel approach that makes it possible to identify slow ring flips in previously inaccessible cases where only single peaks are observed. We demonstrate that Y21 in the bovine pancreatic trypsin inhibitor (BPTI) has a slow ring-flip rate, kflip < 100 s(-1), a result that contrasts with previous estimates of 10(4)-10(6) s(-1) inferred from the single-peak spectrum of Y21. Comparison with a recent 1 ms molecular dynamics trajectory of BPTI shows qualitative agreement and highlights the value of accurate aromatic ring flip data as an important benchmark for molecular dynamics simulations of proteins across wide time scales.

Site-selective 13C labeling of proteins using erythrose

“…For µs (Weininger et al 2014a) and ms (Weininger et al 2012b) dynamics studies however deuteration is usually not required, but serves to prevent rather uncommon strong-coupling effects, which complicate recorded relaxation dispersions, but also contain additional information. Based on the dependence of strong-coupling effects on the refocusing frequency, slow ring-flips for degenerate chemical shifts could be identified (Weininger et al 2013). Further, deuteration is not needed in order to get improved spectra for structural studies (Milbradt et al 2015) or studies involving residual dipolar couplings (RDCs) (Sathyamoorthy et al 2013).…”

Dynamics of Hydrophobic Core Phenylalanine Residues Probed by Solid-State Deuteron NMR

“…13, 14 Solid state offers additional approaches for the characterization of motions in aromatic side chains on slow timescales due to the absence of overall molecular tumbling in the solid phase. In solution, overall molecular tumbling can often complicate the observation of motional modes on timescales close to or slower than those of the tumbling.…”

Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

“…The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al in J Phys Chem B 117: 9241–9247, 2013b) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling.…”

“…Transitions between different conformations that lead to modulation of NMR parameters, such as the chemical shift (Gutowsky and Saika 1953) or residual dipolar couplings (Igumenova et al 2007; Vallurupalli et al 2007), result in exchange contributions to the transverse relaxation rate, which can be probed by NMR relaxation dispersion methods to yield unique information on the structures, thermodynamics and dynamics of the underlying process (Palmer et al 2001; Akke 2002). Experiments have been designed to probe conformational exchange at specific sites in proteins, including the backbone (Akke and Palmer 1996; Ishima et al 1998, 2004; Loria et al 1999a, b; Hill et al 2000; Mulder and Akke 2003; Lundström and Akke 2005a, b; Igumenova and Palmer 2006; Lundström et al 2009a) and side-chain aliphatic (Lundström et al 2007b, 2009b; Hansen et al 2012), methyl (Ishima et al 1999; Mulder et al 2002; Brath et al 2006; Lundström et al 2007b; Weininger et al 2012b, 2013a), carbonyl/carboxyl (Paquin et al 2008; Hansen and Kay 2011) and recently also aromatic groups (Teilum et al 2006; Weininger et al 2012c, 2013b). …”

“…Therefore, aromatic residues are useful probes of the dynamics of the hydrophobic core. Indeed, studies of aromatic ring dynamics have a very long history in NMR, dating back to the 1970s when aromatic ring flips were first observed in proteins (Wüthrich and Wagner 1975; Campbell et al 1975; Hull and Sykes 1975; Wagner et al 1976) and has recently seen a renaissance (Skalicky et al 2001; Sathyamoorthy et al 2013; Weininger et al 2013b; Kasinath et al 2013). …”