Assessing the applicability of ¹⁹F labeled tryptophan residues to quantify protein dynamics

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes

Observation of conformational changes that underlie the catalytic cycle of Xrn2

Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5′→3′ exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information

Multi‐Site Conformational Exchange in the Synthetic Neomycin‐Sensing Riboswitch Studied by 19 F NMR

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19F NMR methods to characterize complex exchange processes with multiple excited states

Methyl TROSY spectroscopy: A versatile NMR approach to study challenging biological systems

A major goal in structural biology is to unravel how molecular machines function in detail. To that end, solution-state NMR spectroscopy is ideally suited as it is able to study biological assemblies in a near natural environment. Based on methyl TROSY methods, it is now possible to record high-quality data on complexes that are far over 100 kDa in molecular weight. In this review, we discuss the theoretical background of methyl TROSY spectroscopy, the information that can be extracted from methyl TROSY spectra and approaches that can be used to assign methyl resonances in large complexes. In addition, we touch upon insights that have been obtained for a number of challenging biological systems, including the 20S proteasome, the RNA exosome, molecular chaperones and G-protein-coupled receptors. We anticipate that methyl TROSY methods will be increasingly important in modern structural biology approaches, where information regarding static structures is complemented with insights into conformational changes and dynamic intermolecular interactions. (C) 2019 The Author(s). Published by Elsevier B.V

Dcp2: an mRNA decapping enzyme that adopts many different shapes and forms

Eukaryotic mRNAs contain a 5' cap structure that protects the transcript against rapid exonucleolytic degradation. The regulation of cellular mRNA levels therefore depends on a precise control of the mRNA decapping pathways. The major mRNA decapping enzyme in eukaryotic cells is Dcp2. It is regulated by interactions with several activators, including Dcp1, Edc1, and Edc3, as well as by an autoinhibition mechanism. The structural and mechanistical characterization of Dcp2 complexes has long been impeded by the high flexibility and dynamic nature of the enzyme. Here we review recent insights into the catalytically active conformation of the mRNA decapping complex, the mode of action of decapping activators and the large interactions network that Dcp2 is embedded in

A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping

Cellular liquid-liquid phase separation (LLPS) results in the formation of dynamic granules that play an important role in many biological processes. On a molecular level, the clustering of proteins into a confined space results from an indefinite network of intermolecular interactions. Here, we introduce and exploit a novel high-throughput bottom-up approach to study how the interactions between RNA, the Dcp1:Dcp2 mRNA decapping complex and the scaffolding proteins Edc3 and Pdc1 result in the formation of processing bodies. We find that the LLPS boundaries are close to physiological concentrations upon inclusion of multiple proteins and RNA. Within in vitro processing bodies the RNA is protected against endonucleolytic cleavage and the mRNA decapping activity is reduced, which argues for a role of processing bodies in temporary mRNA storage. Interestingly, the intrinsically disordered region (IDR) in the Edc3 protein emerges as a central hub for interactions with both RNA and mRNA decapping factors. In addition, the Edc3 IDR plays a role in the formation of irreversible protein aggregates that are potentially detrimental for cellular homeostasis. In summary, our data reveal insights into the mechanisms that lead to cellular LLPS and into the way this influences enzymatic activity

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of F-19-based CPMG, on-resonance R-1 rho and off-resonance R-1 rho RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the F-19 RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with F-19 RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of F-19 NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions