Petra Rovó

Sequential backbone assignment based on dipolar amide-to-amide correlation experiments.

Abstract Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common 13C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR.

Access to aliphatic protons as reporters in non-deuterated proteins by solid-state NMR

Interactions within proteins, with their surrounding, and with other molecules are mediated mostly by hydrogen atoms. In fully protonated, inhomogeneous, or larger proteins, however, aliphatic proton shifts tend to show little dispersion despite fast Magic-Angle Spinning. 3D correlations dispersing aliphatic proton shifts by their better resolved amide N/H shifts can alleviate this problem. Using inverse second-order cross-polarization (iSOCP), we here introduce dedicated and improved means to sensitively link site-specific chemical shift information from aliphatic protons with a backbone amide resolution. Thus, even in cases where protein deuteration is impossible, this approach may enable access to various aspects of protein functions that are reported on by protons.

Protein conformational dynamics studied by 15N and 1H R1ρ relaxation dispersion: Application to wild-type and G53A ubiquitin crystals

Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45-60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use 15N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-1H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of 1H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.

Microsecond Time Scale Proton Rotating-Frame Relaxation under Magic Angle Spinning

This paper deals with the theoretical foundation of proton magic angle spinning rotating-frame relaxation (R1ρ) and establishes the range of validity and accuracy of the presented approach to describe low-amplitude microsecond time scale motion in the solid state. Beside heteronuclear dipolar and chemical shift anisotropy interactions, a major source of relaxation for protons is the homonuclear dipolar interaction. For this latter relaxation process, no general analytical equation has been published until now, which would describe the R1ρ relaxation at any spinning speed, spin-lock field, or tilt angle. To validate the derived equations, we compared the analytical relaxation rates, obtained by solving the master equation within the framework of Redfield theory, with numerically simulated relaxation rates. We found that for small opening angles (∼10°), the relaxation rates obtained with stochastic Liouville simulations agree well with the analytical Redfield relaxation rates for a large range of motional correlation times. However, deviations around the rotary-resonance conditions highlight the fact that Redfield treatment of the solid-state relaxation rates can only provide qualitative insights into the microsecond time scale motion.

The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility.

Significance Sterol regulatory element-binding protein (SREBP) signaling is responsible for transcriptional regulation of cellular lipid homeostasis. Aberrant regulation of the feedback loop that switches on transcription of related genes upon depletion of fatty acids and cholesterol abrogates cellular integrity and is a hallmark of many cancers and other diseases. Regulated intramembrane proteolysis is a conserved mechanism also involved in other signaling processes like Wnt and Notch and defines cellular regulation from homeostasis to proliferation, compartmentalization, and differentiation. An understanding of basic features, however, such as substrate selectivity and specificity, has remained elusive to date. Understanding the proteolysis of the SREBP precursor is an important goal both for understanding and medically intervening in SREBP signaling and related diseases as well as for understanding other regulated intramembrane proteolysis-dependent processes. Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9–10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment.

Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins

Solid-state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic-resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long-distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton-proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin-labeled 1 mg protein samples using fast magic angle spinning.

Microsecond timescale protein dynamics: a combined solid-state NMR approach

Conformational exchange in proteins is a major determinant in protein functionality. In particular, the μs-ms timescale is associated with enzymatic activity and interactions between biological molecules. We show here that a comprehensive data set of R1ρ relaxation dispersion profiles employing multiple effective fields and tilt angles can be easily obtained in perdeuterated, partly back-exchanged proteins at fast magic-angle spinning and further complemented with chemical-exchange saturation transfer NMR experiments. The approach exploits complementary sources of information and enables the extraction of multiple exchange parameters for μs-ms timescale conformational exchange, most notably including the sign of the chemical shift differences between the ground and excited states.

Four faces of the interaction between ions and aromatic rings

Non‐covalent interactions between ions and aromatic rings play an important role in the stabilization of macromolecular complexes; of particular interest are peptides and proteins containing aromatic side chains (Phe, Trp, and Tyr) interacting with negatively (Asp and Glu) and positively (Arg and Lys) charged amino acid residues. The structures of the ion–aromatic‐ring complexes are the result of an interaction between the large quadrupole moment of the ring and the charge of the ion. Four attractive interaction types are proposed to be distinguished based on the position of the ion with respect to the plane of the ring: perpendicular cation–π (CP⊥), co‐planar cation–π (CP∥), perpendicular anion–π (AP⊥), and co‐planar anion–π (AP∥). To understand more than the basic features of these four interaction types, a systematic, high‐level quantum chemical study is performed, using the X– + C6H6, M+ + C6H6, X– + C6F6, and M+ + C6F6 model systems with X− = H−, F−, Cl−, HCOO−, CH3COO− and M+ = H+, Li+, Na+, NH4+ , CH3 NH3+ , whereby C6H6 and C6F6 represent an electron‐rich and an electron‐deficient π system, respectively. Benchmark‐quality interaction energies with small uncertainties, obtained via the so‐called focal‐point analysis (FPA) technique, are reported for the four interaction types. The computations reveal that the interactions lead to significant stabilization, and that the interaction energy order, given in kcal mol−1 in parentheses, is CP⊥ (23–37) > AP⊥ (14–21) > CP∥ (9–22) > AP∥ (6–16). A natural bond orbital analysis performed leads to a deeper qualitative understanding of the four interaction types. To facilitate the future quantum chemical characterization of ion–aromatic‐ring interactions in large biomolecules, the performance of three density functional theory methods, B3LYP, BHandHLYP, and M06‐2X, is tested against the FPA benchmarks, with the result that the M06‐2X functional performs best.

Dynamics and Interactions of a 29 kDa Human Enzyme Studied by Solid-State NMR

Solid-state NMR has been employed for characterization of a broad range of biomacromolecules and supramolecular assemblies. However, because of limitations in sensitivity and resolution, the size of the individual monomeric units has rarely exceeded 15 kDa. As such, enzymes, which are often more complex and comprise long peptide chains, have not been easily accessible, even though manifold desirable information could potentially be provided by solid-state NMR studies. Here, we demonstrate that more than 1200 backbone and side-chain chemical shifts can be reliably assessed from minimal sample quantities for a 29 kDa human enzyme of the carbonic anhydrase family, giving access to its backbone dynamics and intermolecular interactions with a small-molecule inhibitor. The possibility of comprehensive assessment of enzymes in this molecular-weight regime without molecular-tumbling-derived limitations enables the study of residue-specific properties important for their mode of action as well as for pharmacological interference in this and many other enzymes.

Protons as Versatile Reporters in Solid-State NMR Spectroscopy

Solid-state nuclear magnetic resonance (ssNMR) is a spectroscopic technique that is used for characterization of molecular properties in the solid phase at atomic resolution. In particular, using the approach of magic-angle spinning (MAS), ssNMR has seen widespread applications for topics ranging from material sciences to catalysis, metabolomics, and structural biology, where both isotropic and anisotropic parameters can be exploited for a detailed assessment of molecular properties. High-resolution detection of protons long represented the holy grail of the field. With its high natural abundance and high gyromagnetic ratio, 1H has naturally been the most important nucleus type for the solution counterpart of NMR spectroscopy. In the solid state, similar benefits are obtained over detection of heteronuclei, however, a rocky road led to its success as their high gyromagnetic ratio has also been associated with various detrimental effects. Two exciting approaches have been developed in recent years that enable proton detection: After partial deuteration of the sample to reduce the proton spin density, the exploitation of protons could begin. Also, faster MAS, nowadays using tiny rotors with frequencies up to 130 kHz, has relieved the need for expensive deuteration. Apart from the sheer gain in sensitivity from choosing protons as the detection nucleus, the proton chemical shift and several other useful aspects of protons have revolutionized the field. In this Account, we are describing the fundamentals of proton detection as well as the arising possibilities for characterization of biomolecules as associated with the developments in our own lab. In particular, we focus on facilitated chemical-shift assignment, structure calculation based on protons, and on assessment of dynamics in solid proteins. For example, the proton chemical-shift dimension adds additional information for resonance assignments in the protein backbone and side chains. Chemical shifts and high gyromagnetic ratio of protons enable direct readout of spatial information over large distances. Dynamics in the protein backbone or side chains can be characterized efficiently using protons as reporters. For all of this, the sample amounts necessary for a given signal-to-noise have drastically shrunk, and new methodology enables assessment of molecules with increasing monomer molecular weight and complexity. Taken together, protons are able to overcome previous limitations, by speeding up processes, enhancing accuracies, and increasing the accessible ranges of ssNMR spectroscopy, as we shall discuss in detail in the following. In particular, these methodological developments have been pushing solid-state NMR into a new regime of biological topics as they realistically allow access to complex cellular molecules, elucidating their functions and interactions in a multitude of ways.