Ring Flips Revisited: ¹³C Relaxation Dispersion Measurements of Aromatic Side Chain Dynamics and Activation Barriers in Basic Pancreatic Trypsin Inhibitor

Abstract

Intramolecular motions of proteins are critical for biological function. Transient structural fluctuations underlie a wide range of processes, including enzyme catalysis, ligand binding to buried sites, and generic protein motions, such as 180° rotation of aromatic side chains in the protein interior, but remain poorly understood. Understanding the dynamics and molecular nature of concerted motions requires characterization of their rates and energy barriers. Here we use recently developed (13)C transverse relaxation dispersion methods to improve our current understanding of aromatic ring flips in basic pancreatic trypsin inhibitor (BPTI). We validate these methods by benchmarking ring-flip rates against the three previously characterized cases in BPTI, namely, Y23, Y35, and F45. Further, we measure conformational exchange for one additional aromatic ring, F22, which can be interpreted in terms of a flip rate of 666 s(-1) at 5 °C. Upon inclusion of our previously reported result that Y21 also flips slowly [Weininger, U., et al. (2013) J. Phys. Chem. B 117, 9241-9247], the (13)C relaxation dispersion experiments thus reveal relatively slow ring-flip rates for five of eight aromatic residues in BPTI. These results are in contrast with previous reports, which have estimated that all rings, except Y23, Y35, and F45, flip with a high rate at ambient temperature. The (13)C relaxation dispersion data result in an updated rank order of ring-flip rates in BPTI, which agrees considerably better with that estimated from a recent 1 ms molecular dynamics trajectory than do previously published NMR data. However, significant quantitative differences remain between experiment and simulation, in that the latter yields flip rates that are in many cases too fast by 1-2 orders of magnitude. By measuring flip rates across a temperature range of 5-65 °C, we determined the activation barriers of ring flips for Y23, Y35, and F45. Y23 and F45 have identical activation parameters, suggesting that the fluctuations of the protein core around these residues are similar in character. Y35 differs from the other two in its apparent activation entropy. These results might be rationalized by the fact that Y23 and F45 are located in the same region of the structure while Y35 is remote from the other two rings. As indicated by our new results for the exceptionally well-characterized protein BPTI, (13)C relaxation dispersion experiments open the possibility of studying ring flips in a range of cases wider than that previously possible.

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

“…However, in the Anton simulation, we observed the transition to the excited state between 18.6 ms and 27 ms, suggesting a transition timescale two orders of magnitude faster than found in experiment. Although only a single transition in one single long-timescale simulation was observed, and therefore no determination of the timescale for the transition can be made, the order-of-magnitude difference between our observed transition and the experimental rate is consistent with previously reported discrepancies between simulation and experiment for slow ring flips (on the order of microseconds) in proteins (55,56). The fortuitous observation of this transition is consistent with force-field-induced biases toward greater helical content (48) that could have led to the faster transition timescale, since refolding of the F and G helices into a single helix is a key structural change on the path from the ground state to the excited state of L99A.…”

Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO

“…Errors in the extracted R 2eff were estimated from the signal-to-noise ratio using error propagation. The relaxation dispersion data were analyzed using CPMGfit v2.3 (38). Relaxation dispersion curves were fitted to the Carver-Richards (39) two-state exchange model (40),…”

Site-selective 13C labeling of proteins using erythrose

“…While the majority of studies have focused on the protein backbone using inexpensive and robust 15 N labeling (Akke and Palmer 1996; Ishima and Torchia 2003; Jarymowycz and Stone 2006; Loria et al 1999), more and more methods have been developed to study amino-acid side chains (Hansen and Kay 2011; Hansen et al 2012; Lundstrom et al 2009; Millet et al 2002; Muhandiram et al 1995; Mulder et al 2002; Paquin et al 2008). These approaches complement existing backbone studies and widen the view on certain processes, but also enable unique additional information of structure (Korzhnev et al 2010; Neudecker et al 2012), ring-flips (Weininger et al 2014b), and proton occupancy and transfer reactions (Hansen and Kay 2014; Wallerstein et al 2015). A key requirement therefore is to site-selectively label the protein, in order to generate isolated 1 H- 13 C spin pairs (for fast dynamics also isolated 2 H) that are not affected by coupling with their neighbours.…”

“…Experiments designed to characterize dynamics on the ms (Weininger et al 2012b) and µs (Weininger et al 2014a) time-scales have been developed. We have recently reinvestigated the ring-flips in BPTI (Weininger et al 2014b) using these methods, which enabled us to resolve inconsistencies between experiments (Wagner et al 1987, 1976) and molecular dynamics simulations (Shaw et al 2010). …”