Publications by authors named "Petra Rovó"

25 Publications

  • Page 1 of 1

Hydration-Induced Structural Transitions in Biomimetic Tandem Repeat Proteins.

J Phys Chem B 2021 03 17;125(8):2134-2145. Epub 2021 Feb 17.

Faculty of Chemistry and Pharmacy, Department of Chemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany.

A major challenge in developing biomimetic, high-performance, and sustainable products is the accurate replication of the biological materials' striking properties, such as high strength, self-repair, and stimuli-responsiveness. The rationalization of such features on the microscopic scale, together with the rational design of synthetic materials, is currently hindered by our limited understanding of the sequence-structure-property relationship. Here, employing state-of-the-art nuclear magnetic resonance (NMR) spectroscopy, we link the atomistic structural and dynamic properties of an artificial bioinspired tandem repeat protein TR(1,11) to its stunning macroscopic properties including high elasticity, self-healing capabilities, and record-holding proton conductivity among biological materials. We show that the hydration-induced structural rearrangement of the amorphous Gly-rich soft segment and the ordered Ala-rich hard segment is the key to the material's outstanding physical properties. We found that in the hydrated state both the Ala-rich ordered and Gly-rich disordered parts contribute to the formation of the nanoconfined β-sheets, thereby enhancing the strength and toughness of the material. This restructuring is accompanied by fast proline ring puckering and backbone - isomerization at the water-protein interface, which in turn enhances the elasticity and the thermal conductivity of the hydrated films. Our in-depth characterization provides a solid ground for the development of next-generation materials with improved properties.
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http://dx.doi.org/10.1021/acs.jpcb.0c11505DOI Listing
March 2021

Small molecule induced toxic human-IAPP species characterized by NMR.

Chem Commun (Camb) 2020 Nov 2;56(86):13129-13132. Epub 2020 Oct 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.
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http://dx.doi.org/10.1039/d0cc04803hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641245PMC
November 2020

Impact of 5-formylcytosine on the melting kinetics of DNA by 1H NMR chemical exchange.

Nucleic Acids Res 2020 09;48(15):8796-8807

Faculty of Chemistry and Pharmacy, Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany.

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.
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http://dx.doi.org/10.1093/nar/gkaa589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470965PMC
September 2020

Protein Motional Details Revealed by Complementary Structural Biology Techniques.

Structure 2020 09 23;28(9):1024-1034.e3. Epub 2020 Jun 23.

Faculty for Chemistry and Pharmacy, Ludwig-Maximilians-University Munich, 81377 Munich, Germany; Faculty of Chemistry and Chemical Biology, Technical University Dortmund, 44227 Dortmund, Germany. Electronic address:

Proteins depend on defined molecular plasticity for their functionality. How to comprehensively capture dynamics correctly is of ubiquitous biological importance. Approaches commonly used to probe protein dynamics include model-free elucidation of site-specific motion by NMR relaxation, molecular dynamics (MD)-based approaches, and capturing the substates within a dynamic ensemble by recent eNOE-based multiple-structure approaches. Even though MD is sometimes combined with ensemble-averaged NMR restraints, these approaches have largely been developed and used individually. Owing to the different underlying concepts and practical requirements, it has remained unclear how they compare, and how they cross-validate and complement each other. Here, we extract and compare the differential information contents of MD simulations, NMR relaxation measurements, and eNOE-based multi-state structures for the SH3 domain of chicken α-spectrin. The data show that a validated, consistent, and detailed picture is feasible both for timescales and actual conformational states sampled in the dynamic ensemble. This includes the biologically important side-chain plasticity, for which experimentally cross-validated assessment is a significant challenge.
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http://dx.doi.org/10.1016/j.str.2020.06.001DOI Listing
September 2020

Recent advances in solid-state relaxation dispersion techniques.

Authors:
Petra Rovó

Solid State Nucl Magn Reson 2020 08 20;108:101665. Epub 2020 May 20.

Department of Chemistry, Ludwig Maximilian University Munich, Butenandtstr. 5-13, 81377, Munich, Germany; Center for NanoScience (CeNS), Schellingstr. 4, 80799, Munich, Germany. Electronic address:

This review describes two rotating-frame (R) relaxation dispersion methods, namely the Bloch-McConnell Relaxation Dispersion and the Near-rotary Resonance Relaxation Dispersion, which enable the study of microsecond time-scale conformational fluctuations in the solid state using magic-angle-spinning nuclear magnetic resonance spectroscopy. The goal is to provide the reader with key ideas, experimental descriptions, and practical considerations associated with R measurements that are needed for analyzing relaxation dispersion and quantifying conformational exchange. While the focus is on protein motion, many presented concepts can be equally well adapted to study the microsecond time-scale dynamics of other bio- (e.g. lipids, polysaccharides, nucleic acids), organic (e.g. pharmaceutical compounds), or inorganic molecules (e.g., metal organic frameworks). This article summarizes the essential contributions made by recent theoretical and experimental solid-state NMR studies to our understanding of protein motion. Here we discuss recent advances in fast MAS applications that enable the observation and atomic level characterization of sparsely populated conformational states which are otherwise inaccessible for other experimental methods. Such high-energy states are often associated with protein functions such as molecular recognition, ligand binding, or enzymatic catalysis, as well as with disease-related properties such as misfolding and amyloid formation.
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http://dx.doi.org/10.1016/j.ssnmr.2020.101665DOI Listing
August 2020

Rational Design of Covalent Cobaloxime-Covalent Organic Framework Hybrids for Enhanced Photocatalytic Hydrogen Evolution.

J Am Chem Soc 2020 07 6;142(28):12146-12156. Epub 2020 Jul 6.

Max Planck Institute for Solid State Research, Heisenbergstrasse 1, 70569 Stuttgart, Germany.

Covalent organic frameworks (COFs) display a unique combination of chemical tunability, structural diversity, high porosity, nanoscale regularity, and thermal stability. Recent efforts are directed at using such frameworks as tunable scaffolds for chemical reactions. In particular, COFs have emerged as viable platforms for mimicking natural photosynthesis. However, there is an indisputable need for efficient, stable, and economical alternatives for the traditional platinum-based cocatalysts for light-driven hydrogen evolution. Here, we present azide-functionalized chloro(pyridine)cobaloxime hydrogen-evolution cocatalysts immobilized on a hydrazone-based COF-42 backbone that show improved and prolonged photocatalytic activity with respect to equivalent physisorbed systems. Advanced solid-state NMR and quantum-chemical methods allow us to elucidate details of the improved photoreactivity and the structural composition of the involved active site. We found that a genuine interaction between the COF backbone and the cobaloxime facilitates recoordination of the cocatalyst during the photoreaction, thereby improving the reactivity and hindering degradation of the catalyst. The excellent stability and prolonged reactivity make the herein reported cobaloxime-tethered COF materials promising hydrogen evolution catalysts for future solar fuel technologies.
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http://dx.doi.org/10.1021/jacs.0c02155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366382PMC
July 2020

Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy.

J Am Chem Soc 2019 12 5;141(49):19276-19288. Epub 2019 Nov 5.

Faculty of Chemistry and Chemical Biology , Technical University Dortmund , Otto-Hahn-Straße 4a , 44227 Dortmund , Germany.

Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many biomacromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water sites can be easily detected by X-ray crystallography, the dynamics within functional water-protein network architectures is largely elusive. Here we use solid-state NMR relaxation dispersion measurements with a focus on those active-site residues in the enzyme human carbonic anhydrase II (hCAII) that constitute the evolutionarily conserved water pocket, key for CAs' enzymatic catalysis. Together with chemical shifts, peak broadening, and results of molecular dynamics (MD) and DFT shift calculations, the relaxation dispersion data suggest the presence of a widespread fast μs-time-scale dynamics in the pocket throughout the protein-water network. This process is abrogated in the presence of an inhibitor which partially disrupts the network. The time scale of the protein-water pocket motion coincides both with the estimated residence time of Zn-bound water/OH in the pocket showing the longest lifetimes in earlier magnetic relaxation dispersion experiments as well as with the rate-limiting step of catalytic turnover. As such, the reorganization of the water pocket:enzyme architecture might constitute an element of importance for enzymatic activity of this and possibly other proteins.
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http://dx.doi.org/10.1021/jacs.9b05311DOI Listing
December 2019

Proto-Urea-RNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides.

Angew Chem Int Ed Engl 2019 12 30;58(51):18691-18696. Epub 2019 Oct 30.

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU München, Butenandtstr. 5-13, 81377, München, Germany.

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information-carrying RNA oligomers able to self-replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto-RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis-urea (biuret) and tris-urea (triuret) are able to directly react with ribose. The urea-ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides' base-pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea-containing RNA bases are good candidates for a proto-RNA world.
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http://dx.doi.org/10.1002/anie.201911746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916321PMC
December 2019

Mechanistic Insights into Microsecond Time-Scale Motion of Solid Proteins Using Complementary N and H Relaxation Dispersion Techniques.

J Am Chem Soc 2019 01 8;141(2):858-869. Epub 2019 Jan 8.

Department Chemie und Pharmazie , Ludwig-Maximilians-Universität München , 81377 München , Germany.

NMR relaxation dispersion methods provide a holistic way to observe microsecond time-scale protein backbone motion both in solution and in the solid state. Different nuclei (H and N) and different relaxation dispersion techniques (Bloch-McConnell and near-rotary-resonance) give complementary information about the amplitudes and time scales of the conformational dynamics and provide comprehensive insights into the mechanistic details of the structural rearrangements. In this paper, we exemplify the benefits of the combination of various solution- and solid-state relaxation dispersion methods on a microcrystalline protein (α-spectrin SH3 domain), for which we are able to identify and model the functionally relevant conformational rearrangements around the ligand recognition loop occurring on multiple microsecond time scales. The observed loop motions suggest that the SH3 domain exists in a binding-competent conformation in dynamic equilibrium with a sterically impaired ground-state conformation both in solution and in crystalline form. This inherent plasticity between the interconverting macrostates is compatible with a conformational-preselection model and provides new insights into the recognition mechanisms of SH3 domains.
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http://dx.doi.org/10.1021/jacs.8b09258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982537PMC
January 2019

Protons as Versatile Reporters in Solid-State NMR Spectroscopy.

Acc Chem Res 2018 06 15;51(6):1386-1395. Epub 2018 May 15.

Department Chemistry , Ludwig-Maximilians-University Munich , Butenandtstr. 5-13 , 81377 Munich , Germany.

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, H 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.
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http://dx.doi.org/10.1021/acs.accounts.8b00055DOI Listing
June 2018

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

J Phys Chem Lett 2018 Mar 2;9(6):1307-1311. Epub 2018 Mar 2.

Department Chemistry and Pharmacy , Ludwig-Maximilians-University Munich , Butenandtstr. 5-13 , 81377 Munich , Germany.

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.
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http://dx.doi.org/10.1021/acs.jpclett.8b00110DOI Listing
March 2018

Microsecond Timescale Protein Dynamics: a Combined Solid-State NMR Approach.

Chemphyschem 2018 Jan 14;19(1):34-39. Epub 2017 Dec 14.

Department Chemie und Pharmazie, Ludwig-Maximailians-Universität München, 81377, München, Germany.

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.
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http://dx.doi.org/10.1002/cphc.201701238DOI Listing
January 2018

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

J Phys Chem B 2017 06 14;121(25):6117-6130. Epub 2017 Jun 14.

Department Chemie und Pharmazie, Ludwig-Maximilians-Universität München , 81377 München, Germany.

This paper deals with the theoretical foundation of proton magic angle spinning rotating-frame relaxation (R) 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 R 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.
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http://dx.doi.org/10.1021/acs.jpcb.7b03333DOI Listing
June 2017

Four faces of the interaction between ions and aromatic rings.

J Comput Chem 2017 07 10;38(20):1762-1773. Epub 2017 May 10.

MTA-ELTE Protein Modeling Research Group, Institute of Chemistry, Eötvös Loránd University, H-1518 Budapest 112, P.O. Box 32, Hungary.

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  + C H , M  + C H , X  + C F , and M  + C F model systems with X  = H , F , Cl , HCOO , CH COO and M  = H , Li , Na , NH4+, CH NH3+, whereby C H and C F 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 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. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcc.24816DOI Listing
July 2017

Protein conformational dynamics studied by N and H R relaxation dispersion: Application to wild-type and G53A ubiquitin crystals.

Solid State Nucl Magn Reson 2017 10 14;87:86-95. Epub 2017 Apr 14.

Université Grenoble Alpes, IBS, F-38044 Grenoble, France; CEA, Institut de Biologie Structurale, F-38044 Grenoble, France; CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France. Electronic address:

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 N R 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-H R relaxation to obtain insight into dynamics. We show that while quantitative interpretation of H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.
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http://dx.doi.org/10.1016/j.ssnmr.2017.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531261PMC
October 2017

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

Phys Chem Chem Phys 2016 Mar;18(12):8359-63

Max-Planck Institute for Biophysical Chemistry, Department NMR-Based Structural Biology, Am Fassberg 11, 37077 Göttingen, Germany.

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.
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http://dx.doi.org/10.1039/c5cp06601hDOI Listing
March 2016

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

Proc Natl Acad Sci U S A 2015 Oct 21;112(40):12390-5. Epub 2015 Sep 21.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;

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.
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http://dx.doi.org/10.1073/pnas.1513782112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603520PMC
October 2015

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

Chemphyschem 2015 Dec 1;16(18):3791-6. Epub 2015 Oct 1.

Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

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.
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http://dx.doi.org/10.1002/cphc.201500799DOI Listing
December 2015

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

J Biomol NMR 2015 Jul 15;62(3):303-11. Epub 2015 May 15.

Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

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 (13)C-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.
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http://dx.doi.org/10.1007/s10858-015-9945-4DOI Listing
July 2015

Phosphorylation as conformational switch from the native to amyloid state: Trp-cage as a protein aggregation model.

J Phys Chem B 2015 Feb 6;119(7):2946-55. Epub 2015 Feb 6.

Department of Biochemistry, ‡MTA-ELTE NAP B Neuroimmunology Research Group, and §Department of Anatomy, Cell and Developmental Biology, Institute of Biology Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest, H-1117 Hungary.

The 20 residue long Trp-cage miniprotein is an excellent model for both computational and experimental studies of protein folding and stability. Recently, great attention emerged to study disease-related protein misfolding, aggregation, and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects. It aggregates with ease upon structure destabilization, and thus it is suitable for modeling aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, molecular dynamics (MD) simulations, and transmission electron microscopy (TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant, which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form that can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.
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http://dx.doi.org/10.1021/jp5124234DOI Listing
February 2015

Rational design of α-helix-stabilized exendin-4 analogues.

Biochemistry 2014 Jun 28;53(22):3540-52. Epub 2014 May 28.

Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University , Budapest, Hungary.

Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule's poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both (15)N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.
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http://dx.doi.org/10.1021/bi500033cDOI Listing
June 2014

Structural insights into the Trp-cage folding intermediate formation.

Chemistry 2013 Feb 14;19(8):2628-40. Epub 2013 Jan 14.

Laboratory of Structural Chemistry and Biology, Institute of Chemistry and Protein Modeling Group of HAS-ELTE, Eötvös Loránd University, 1117 Budapest, Pázmány Péter sétány 1/A, Hungary.

The 20 residue long Trp-cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the (15)N and (13)C/(15)N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible--fully folded, unfolded and intermediate--fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH(F-I), T(m)(F-I), ΔC(p)(F-I) and ΔH(I-U), T(m)(I-U), ΔC(p)(I-U)) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp-cage distinct intermediates evolve: a fast-exchanging intermediate is present under neutral conditions, whereas a slow-exchanging intermediate-pair emerges at acidic pH. The fast-exchanging intermediate has a native-like structure with a short α-helix in the G(11)-G(15) segment, whereas the slow-exchanging intermediate-pair presents elevated dynamics, with no detectable native-like residue contacts in which the G(11)-P(12) peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.
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http://dx.doi.org/10.1002/chem.201203764DOI Listing
February 2013

Cooperativity network of Trp-cage miniproteins: probing salt-bridges.

J Pept Sci 2011 Sep 6;17(9):610-9. Epub 2011 Jun 6.

Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös University, Budapest, Hungary.

Trp-cage miniprotein was used to investigate the role of a salt-bridge (Asp(9) -Arg(16) ) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side-chains are also part of a dense interaction network composed of electrostatic, H-bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt-bridge with those of the salt-bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp-cage miniproteins is more complex than a simple two-state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt-bridge stabilization; in the miniprotein variants equipped with salt-bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt-bridge deleted mutants. In this molecular framework Arg(16) stabilizes more than Asp(9) does, because of its higher degree of 3D-fold cooperation. In conclusion, the Xxx(9) leftright arrow Xxx(16) salt-bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network.
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http://dx.doi.org/10.1002/psc.1377DOI Listing
September 2011

Signalogs: orthology-based identification of novel signaling pathway components in three metazoans.

PLoS One 2011 May 3;6(5):e19240. Epub 2011 May 3.

Department of Genetics, Eötvös Loránd University, Budapest, Hungary.

Background: Uncovering novel components of signal transduction pathways and their interactions within species is a central task in current biological research. Orthology alignment and functional genomics approaches allow the effective identification of signaling proteins by cross-species data integration. Recently, functional annotation of orthologs was transferred across organisms to predict novel roles for proteins. Despite the wide use of these methods, annotation of complete signaling pathways has not yet been transferred systematically between species.

Principal Findings: Here we introduce the concept of 'signalog' to describe potential novel signaling function of a protein on the basis of the known signaling role(s) of its ortholog(s). To identify signalogs on genomic scale, we systematically transferred signaling pathway annotations among three animal species, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and humans. Using orthology data from InParanoid and signaling pathway information from the SignaLink database, we predict 88 worm, 92 fly, and 73 human novel signaling components. Furthermore, we developed an on-line tool and an interactive orthology network viewer to allow users to predict and visualize components of orthologous pathways. We verified the novelty of the predicted signalogs by literature search and comparison to known pathway annotations. In C. elegans, 6 out of the predicted novel Notch pathway members were validated experimentally. Our approach predicts signaling roles for 19 human orthodisease proteins and 5 known drug targets, and suggests 14 novel drug target candidates.

Conclusions: Orthology-based pathway membership prediction between species enables the identification of novel signaling pathway components that we referred to as signalogs. Signalogs can be used to build a comprehensive signaling network in a given species. Such networks may increase the biomedical utilization of C. elegans and D. melanogaster. In humans, signalogs may identify novel drug targets and new signaling mechanisms for approved drugs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019240PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086880PMC
May 2011

Uniformly curated signaling pathways reveal tissue-specific cross-talks and support drug target discovery.

Bioinformatics 2010 Aug 11;26(16):2042-50. Epub 2010 Jun 11.

Department of Genetics, Eötvös University, Budapest, Hungary.

Motivation: Signaling pathways control a large variety of cellular processes. However, currently, even within the same database signaling pathways are often curated at different levels of detail. This makes comparative and cross-talk analyses difficult.

Results: We present SignaLink, a database containing eight major signaling pathways from Caenorhabditis elegans, Drosophila melanogaster and humans. Based on 170 review and approximately 800 research articles, we have compiled pathways with semi-automatic searches and uniform, well-documented curation rules. We found that in humans any two of the eight pathways can cross-talk. We quantified the possible tissue- and cancer-specific activity of cross-talks and found pathway-specific expression profiles. In addition, we identified 327 proteins relevant for drug target discovery.

Conclusions: We provide a novel resource for comparative and cross-talk analyses of signaling pathways. The identified multi-pathway and tissue-specific cross-talks contribute to the understanding of the signaling complexity in health and disease, and underscore its importance in network-based drug target selection.

Availability: http://SignaLink.org.
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http://dx.doi.org/10.1093/bioinformatics/btq310DOI Listing
August 2010