Information, Coding, and Biological Function: The Dynamics of Life.

In the mid-20th century, two new scientific disciplines emerged forcefully: molecular biology and information-communication theory. At the beginning, cross-fertilization was so deep that the term genetic code was universally accepted for describing the meaning of triplets of mRNA (codons) as amino acids. However, today, such synergy has not taken advantage of the vertiginous advances in the two disciplines and presents more challenges than answers. These challenges not only are of great theoretical relevance but also represent unavoidable milestones for next-generation biology: from personalized genetic therapy and diagnosis to Artificial Life to the production of biologically active proteins. Moreover, the matter is intimately connected to a paradigm shift needed in theoretical biology, pioneered a long time ago, that requires combined contributions from disciplines well beyond the biological realm. The use of information as a conceptual metaphor needs to be turned into quantitative and predictive models that can be tested empirically and integrated in a unified view. Successfully achieving these tasks requires a wide multidisciplinary approach, including Artificial Life researchers, to address such an endeavour.

4-Oxo-2-nonenal-Induced α-Synuclein Oligomers Interact with Membranes in the Cell, Leading to Mitochondrial Fragmentation.

Oxidative stress and formation of cytotoxic oligomers by the natively unfolded protein α-synuclein (α-syn) are both connected to the development of Parkinson's disease. This effect has been linked to lipid peroxidation and membrane disruption, but the specific mechanisms behind these phenomena remain unclear. To address this, we have prepared α-syn oligomers (αSOs) in vitro in the presence of the lipid peroxidation product 4-oxo-2-nonenal and investigated their interaction with live cells using in-cell NMR as well as stimulated emission depletion (STED) super-resolution and confocal microscopy. We find that the αSOs interact strongly with organellar components, leading to strong immobilization of the protein's otherwise flexible C-terminus. STED microscopy reveals that the oligomers localize to small circular structures inside the cell, while confocal microscopy shows mitochondrial fragmentation and association with both late endosome and retromer complex before the SOs interact with mitochondria. Our study provides direct evidence for close contact between cytotoxic α-syn aggregates and membraneous compartments in the cell.

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein.

The intrinsically disordered human protein α-synuclein (αSN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric αSN is cytotoxic, making it important to devise strategies to either prevent oligomer formation and/or inhibit the ensuing toxicity. (-)-epigallocatechin gallate (EGCG) has emerged as a molecular modulator of αSN self-assembly, as it reduces the flexibility of the C-terminal region of αSN in the oligomer and inhibits the oligomer's ability to perturb phospholipid membranes and induce cell death. However, a detailed structural and kinetic characterization of this interaction is still lacking. Here, we use liquid-state NMR spectroscopy to investigate how EGCG interacts with monomeric and oligomeric forms of αSN. We find that EGCG can bind to all parts of monomeric αSN but exhibits highest affinity for the N-terminal region. Monomeric αSN binds ∼54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the αSN association reaction. In contrast, preformed oligomers only bind ∼7 EGCG molecules per protomer, in agreement with the more compact nature of the oligomer compared with the natively unfolded monomer. In previously conducted cell assays, as little as 0.36 EGCG per αSN reduce oligomer toxicity by 50%. Our study thus demonstrates that αSN cytotoxicity can be inhibited by small molecules at concentrations at least an order of magnitude below full binding capacity. We speculate this is due to cooperative binding of protein-stabilized EGCG dimers, which in turn implies synergy between protein association and EGCG dimerization.

How internal cavities destabilize a protein.

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å-3.

The contribution of electrostatics to hydrogen exchange in the unfolded protein state.

Although electrostatics have long been recognized to play an important role in hydrogen exchange (HX) with solvent, the quantitative assessment of its magnitude in the unfolded state has hitherto been lacking. This limits the utility of HX as a quantitative method to study protein stability, folding, and dynamics. Using the intrinsically disordered human protein α-synuclein as a proxy for the unfolded state, we show that a hybrid mean-field approach can effectively compute the electrostatic potential at all backbone amide positions along the chain. From the electrochemical potential, a fourfold reduction in hydroxide concentration near the protein backbone is predicted for the C-terminal domain, a prognosis that is in direct agreement with experimentally derived protection factors from NMR spectroscopy. Thus, impeded HX for the C-terminal region of α-synuclein is not the result of intramolecular hydrogen bonding and/or structure formation.

Lipid Peroxidation Products HNE and ONE Promote and Stabilize Alpha-Synuclein Oligomers by Chemical Modifications.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

CheSPI: chemical shift secondary structure population inference.

NMR chemical shifts (CSs) are delicate reporters of local protein structure, and recent advances in random coil CS (RCCS) prediction and interpretation now offer the compelling prospect of inferring small populations of structure from small deviations from RCCSs. Here, we present CheSPI, a simple and efficient method that provides unbiased and sensitive aggregate measures of local structure and disorder. It is demonstrated that CheSPI can predict even very small amounts of residual structure and robustly delineate subtle differences into four structural classes for intrinsically disordered proteins. For structured regions and proteins, CheSPI provides predictions for up to eight structural classes, which coincide with the well-known DSSP classification. The program is freely available, and can either be invoked from URL www.protein-nmr.org as a web implementation, or run locally from command line as a python program. CheSPI generates comprehensive numeric and graphical output for intuitive annotation and visualization of protein structures. A number of examples are provided.

Sensitive and simplified: a combinatorial acquisition of five distinct 2D constant-time 13C-1H NMR protein correlation spectra.

A procedure is presented for the substantial simplification of 2D constant-time 13C-1H heteronuclear single-quantum correlation (HSQC) spectra of 13C-enriched proteins. In this approach, a single pulse sequence simultaneously records eight sub-spectra wherein the phases of the NMR signals depend on spin topology. Signals from different chemical groups are then stratified into different sub-spectra through linear combination based on Hadamard encoding of 13CHn multiplicity (n = 1, 2, and 3) and the chemical nature of neighboring 13C nuclei (aliphatic, carbonyl/carboxyl, aromatic). This results in five sets of 2D NMR spectra containing mutually exclusive signals from: (i) 13Cß-1Hß correlations of asparagine and aspartic acid, 13Cγ-1Hγ correlations of glutamine and glutamic acid, and 13Cα-1Hα correlations of glycine, (ii) 13Cα-1Hα correlations of all residues but glycine, and (iii) 13Cß-1Hß correlations of phenylalanine, tyrosine, histidine, and tryptophan, and the remaining (iv) aliphatic 13CH2 and (v) aliphatic 13CH/13CH3 resonances. As HSQC is a common element of many NMR experiments, the spectral simplification proposed in this article can be straightforwardly implemented in experiments for resonance assignment and structure determination and should be of widespread utility.

Predicting NMR relaxation of proteins from molecular dynamics simulations with accurate methyl rotation barriers.

The internal dynamics of proteins occurring on time scales from picoseconds to nanoseconds can be sensitively probed by nuclear magnetic resonance (NMR) spin relaxation experiments, as well as by molecular dynamics (MD) simulations. This complementarity offers unique opportunities, provided that the two methods are compared at a suitable level. Recently, several groups have used MD simulations to compute the spectral density of backbone and side chain molecular motions and to predict NMR relaxation rates from these. Unfortunately, in the case of methyl groups in protein side chains, inaccurate energy barriers to methyl rotation were responsible for a systematic discrepancy in the computed relaxation rates, as demonstrated for the AMBER ff99SB*-ILDN force field (and related parameter sets), impairing quantitative agreement between simulations and experiments. However, correspondence could be regained by emending the MD force field with accurate coupled cluster quantum chemical calculations. Spurred by this positive result, we tested whether this approach could be generally applicable, in spite of the fact that different MD force fields employ different water models. Improved methyl group rotation barriers for the CHARMM36 and AMBER ff15ipq protein force fields were derived, such that the NMR relaxation data obtained from the MD simulations even now display very good agreement with the experiment. Results herein showcase the performance of present-day MD force fields and manifest their refined ability to accurately describe internal protein dynamics.

Fuzzy and fast nuclear transport.

Exchange of macromolecules between the cytoplasm and the nucleus of all eukaryotic cells is controlled by nuclear pore complexes, which form a selective permeability barrier. The requirement for rapid but selective transport leads to a "transport paradox." A new experimental study now provides a thermodynamic explanation.

pepKalc: scalable and comprehensive calculation of electrostatic interactions in random coil polypeptides.

Motivation: Polypeptide sequence length is the single dominant factor hampering the effectiveness of currently available software tools for de novo calculation of amino acid-specific protonation constants in disordered polypeptides. Results: We have developed pepKalc, a robust simulation software for the comprehensive evaluation of protein electrostatics in unfolded states. Our software completely removes the limitations of the previously reported Monte-Carlo approaches in the computation of protein electrostatics by using a hybrid approach that effectively combines exact and mean-field calculations to rapidly obtain accurate results. Paired with a modern architecture GPU, pepKalc is capable of evaluating protonation behavior for an arbitrary-size polypeptide in a sub-second time regime. Availability and implementation: http://protein-nmr.org and https://github.com/PeptoneInc/pepkalc.

Measurement of Very Fast Exchange Rates of Individual Amide Protons in Proteins by NMR Spectroscopy.

NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 104  s-1 . Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2Nx Hz by exchange events that occur during a series of pi pulses on the 15 N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D9k , where exchange rates up to 600 s-1 were measured for amides, where no signal was detectable in 15 N-1 H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47-69 kJ mol-1 for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.

Fast and Quantitative NMR Metabolite Analysis Afforded by a Paramagnetic Co-Solute.

NMR spectroscopy is an indispensable technique for the determination of the chemical identity and structure of small molecules. The method is especially recognized for its robustness and intrinsically quantitative nature, and has manifested itself as a key analytical platform for diverse fields of application, ranging from chemical synthesis to metabolomics. Unfortunately, the slow recovery of nuclear spin polarization by spin-lattice (T1 ) relaxation causes most experimental time to be lost on idle waiting. Furthermore, truly quantitative NMR (qNMR) spectroscopy requires waiting times of 5-times the longest T1 in the sample, making qNMR spectroscopy slow and inefficient. We demonstrate here that co-solute paramagnetic relaxation can mitigate these two problems simultaneously. The addition of a small amount of paramagnetic gadolinium chelate, available in the form of commercial contrast-agent solutions, enables cheap, quantitative, and efficient high-throughput mixture analysis.

MOAG-4 promotes the aggregation of α-synuclein by competing with self-protective electrostatic interactions.

Aberrant protein aggregation underlies a variety of age-related neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Little is known, however, about the molecular mechanisms that modulate the aggregation process in the cellular environment. Recently, MOAG-4/SERF has been identified as a class of evolutionarily conserved proteins that positively regulates aggregate formation. Here, by using nuclear magnetic resonance (NMR) spectroscopy, we examine the mechanism of action of MOAG-4 by characterizing its interaction with α-synuclein (α-Syn). NMR chemical shift perturbations demonstrate that a positively charged segment of MOAG-4 forms a transiently populated α-helix that interacts with the negatively charged C terminus of α-Syn. This process interferes with the intramolecular interactions between the N- and C-terminal regions of α-Syn, resulting in the protein populating less compact forms and aggregating more readily. These results provide a compelling example of the complex competition between molecular and cellular factors that protect against protein aggregation and those that promote it.

POTENCI: prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins.

Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections. RCCS predictions with POTENCI show root-mean-square values that are lower by 25-78%, with the largest improvements observed for 1Hα and 13C'. It is demonstrated how POTENCI can be applied to analyze subtle deviations from RCCSs to detect small populations of residual structure in intrinsically disorder proteins that were not discernible before. POTENCI source code is available for download, or can be deployed from the URL http://www.protein-nmr.org .

Narrowing the gap between experimental and computational determination of methyl group dynamics in proteins.

Nuclear magnetic resonance (NMR) spin relaxation has become the mainstay technique to sample protein dynamics at atomic resolution, expanding its repertoire from backbone 15N to side-chain 2H probes. At the same time, molecular dynamics (MD) simulations have become increasingly powerful to study protein dynamics due to steady improvements of physical models, algorithms, and computational power. Good agreement between generalized Lipari-Szabo order parameters derived from experiment and MD simulation has been observed for the backbone dynamics of a number of proteins. However, the agreement for the more dynamic side-chains, as probed by methyl group relaxation, was much worse. Here, we use T4 lysozyme (T4L), a protein with moderate tumbling anisotropy, to showcase a number of improvements that reduce this gap by a combined evaluation of NMR relaxation experiments and MD simulations. By applying a protein force field with accurate methyl group rotation barriers in combination with a solvation model that yields correct protein rotational diffusion times, we find that properly accounting for anisotropic protein tumbling is an important factor to improve the match between NMR and MD in terms of methyl axis order parameters, spectral densities, and relaxation rates. The best agreement with the experimentally measured relaxation rates is obtained by a posteriori fitting the appropriate internal time correlation functions, truncated by anisotropic overall tumbling. In addition, MD simulations led us to account for a hitherto unrealized artifact in deuterium relaxation experiments arising from strong coupling for leucine residues in uniformly 13C-enriched proteins. For T4L, the improved analysis reduced the RMSD between MD and NMR derived methyl axis order parameters from 0.19 to 0.11. At the level of the spectral density functions, the improvements allow us to extract the most accurate parameters that describe protein side-chain dynamics. Further improvement is challenging not only due to force field and sampling limitations in MD, but also due to inherent limitations of the Lipari-Szabo model to capture complex dynamics.

Active-Site pKa Determination for Photoactive Yellow Protein Rationalizes Slow Ground-State Recovery.

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pKa determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow.

Structural basis for RNA-genome recognition during bacteriophage Qß replication.

Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qß replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy.

Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that a protein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a 13 C-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the 15 Nη and 15 Nϵ chemical shifts of an arginine head group are correlated with that of the directly attached 13 Cζ . In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the 15 N-1 H scalar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three Hη atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.