Nmr relaxation studies on the hydrate layer of intrinsically unstructured proteins

Intrinsically unstructured/ disordered proteins (IUPs) exist in a disordered and largely solvent- exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate ( bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class

A szekurin-szeparáz rendszer szerkezeti-funkcionális jellemzése = Structural and functional characterisation of the securin-separase system

A munkaterv azt a célt tűzte ki, hogy a sejtciklusban fontos szerepet játszó szeparáz enzim és rendezetlen inhibítora (szekurin) részletes szerkezeti elemzésén keresztül közelebb jutunk a rendezetlen fehérjék funkciójának jobb megértéséhez. Ezen tervek nagyrészt megvalósultak, elsősorban a humán szekurin klónozását, és részletes szerkezeti jellemzését sikerült megoldanunk, ezen eredményeket két cikkben publikáltuk. Elkészültünk többféle szekurin (S. cerevisiae, D. melanogaster és humán) összehasonlító szerkezeti vizsgálatával is, ezen eredmények publikációja folyamatban van. A szeparáz enzim katalitikus doménjének klónozása sikerült, nagyszámú konstrukció készült és több is jól kifejeződő fehérjét eredményezett, amelyek aktivitását azonban nem tudtuk mérni. A munkát folytatjuk, folyamatban van egyrészt a fehérje különböző körülmények közötti kifejezése, denaturációja és renaturációja, és aktív formában való előállítása, másrészt nemzetközi együttműködésben Prof. Darren Hart-tal (EMBL Grenoble) az ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) könyvtár-szűrési technika alkalmazása oldható és aktivitást mutató szeparáz konstrukció létrehozására. A fehérjék szerkezeti rendezetlenségének vizsgálata eközben számos más területeken is hatékonyan folyt, az OTKA K60694 pályázat támogatásának összesen 19 publikáció jelent meg. | The main goal of the proposed work was the detailed structure-function characterization of separase, a key enzyme in cell-cycle regulation and its intrinsically disordered inhibitor, securin. We planned to clone and isolate the two proteins, characterize their structure and interaction with particular focus on the disordered structural state of securin. We achieved part of this goal, mostly the cloning, expression and structural characterization by NMR of human securin, which was published in two papers. We also completed the comparative structural study of three different securins (S. cerevisiae, D. melanogaster and human), which is under publication. We managed to clone and express several constructs of the catalytic domain of separase, which resulted in soluble forms of the protein, without enzymatic activity, though. We continue this research by varying expression conditions, including denaturation-renaturation cycles to produce active separase, and also by an international collaboration with Prof. Darren Hart-tal (EMBL Grenoble) with the application of the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technique in order to generate enzymatically active separase. Besides studies on the securin-separase system, we have carried out many other lines of productive research on intrinsically disordered proteins with the help of OTKA K60694 grant: we published 19 papers with acknowledging the grant

Rendezetlen fehérjék: a szerkezet-funkció paradigma kiterjesztése = Intrinsically disordered proteins: extension of the structure-function paradigm

Kísérletes eredmények: Rendezetlen chaperonok hatása, a riboszomális fehérjék kettős chaperone hatásának kimutatása. A rendezetlen fehérjék ellenállnak a hideg-denaturációnak. A rendezetlen fehérjéknek egyedi a hidratációja. Rendezetlenség kimutatása liofilizált mintákból. A rendezetlenség korrelál a kromoszómális transzlokációval. Az állványfehérjéknek magas a rendezetlensége. Új NMR technika a rendezetlen fehérjék elemzésére. Az ERD14 NMR jelhozzárendelése és szerkezeti-dinamikai jellemzése. Az androgén receptor rendezetlen régiójának hossza kapcsolatban van a kognitív képességekkel. A rendezetlen fehérjék crowding körülmények között sem tekerednek fel. Elméleti megfigyelések: Kötött állapotban megfigyelhető rendezetlenség (bolyhsosság) elemzése, a domén koncepció kiterjesztése a rendezetlenségre, az amiloidokban mutatkozó rendezetlenség leírása. Bioinformatikai eredmények: A hipertermofil baktériumokban a rendezetlenség aránya alacsony. Az alternatív splicing során kialakuló kettős kódolású szakaszokról átíródó fehérjék rendezetlensége teszi lehetővé az alternatív fehérjék létét. A rendezetlenség közvetlen kapcsolatban van a fehérje funkciójával, így harmadlagos szerkezeti elemnek tekinthető. Predikciós módszer kidolgozása az alternatív splicing során létrejövő fehérje életképességének megállapítására. A poty-vírus VPg sokféle funkcióban vesz részt, köszönhetően szerkezeti flexibilitásának. Egyéb eredmények: Review a rendezetlenség kutatásának új szakaszáról. | Experimental results: Study of the disordered chaperones, the determination of the double chaperone effect of ribosomal proteins, resistance of the IDPs to cold denaturation. Determination of the unique hydration of IDPs. Determination of protein disorder from lyophilized samples. Protein disorder correlates with chromosomal translocation. Scaffold proteins contain a high level of structural disorder. Designing a new NMR method for the study of IDPs. Assignation of NMR signals of ERD14 and its structural-dynamics characterization. The observation that the length of the disordered region of androgen receptor is related to cognitive capabilities. IDPs remain unfolded even under crowding conditions. Theoretical observations: Analysis of disorder in the bound form (fuzziness), extending the domain concept to IDPs, description for disorder in amyloids. Bioniformatical results: Observation of low levels of disorder in hyperthermophil bacteria. Alternative protein forms are able to evolve because of the disordered nature of the transcripts of dual coding sequences, that result from alternative splicing. Disorder is in intimate connection to function, thus it should come under the category of tertiary structural elements. Designing a prediction method to determine the viability of a protein from an alternative splice event. The VPg protein of poty virus possesses many different functions, thanks to its structural flexibility. Other results: Review of the new era of disorder research

Abundance of intrinsic disorder in SV-IV, a multifunctional androgen-dependent protein secreted from rat seminal vesicle

The potent immunomodulatory, anti-inflammatory and procoagulant properties of the
protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have been
previously found to be modulated by a supramolecular monomer-trimer equilibrium.
More structural details that integrate experimental data into a predictive framework
have recently been reported. Unfortunately, homology modelling and fold-recognition
strategies were not successful in creating a theoretical model of the structural
organization of SV-IV. It was inferred that the global structure of SV-IV is not similar
to any protein of known three-dimensional structure. Reversing the classical approach
to the sequence-structure-function paradigm, in this paper we report on novel
information obtained by comparing physicochemical parameters of SV-IV with two
datasets made of intrinsically unfolded and ideally globular proteins. In addition, we
have analysed the SV-IV sequence by several publicly available disorder-oriented
predictors. Overall, disorder predictions and a re-examination of existing experimental
data strongly suggest that SV-IV needs large plasticity to efficiently interact with the
different targets that characterize its multifaceted biological function and should be
therefore better classified as an intrinsically disordered protein

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication

Primary Contact Sites in Intrinsically Unstructured Proteins: The Case of Calpastatin And Microtubule-associated Protein 2

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state H-1 NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.e., subdomains A, B, and C in CSD1 and the proline-rich region (PRR) in MAP2c, that are destined to form contacts with their targets. For CSD1, nonadditivity of the CD spectra of its two halves and suboptimal hydration of the full-length protein measured by solid-state NMR demonstrate that long-range tertiary interactions provide the structural background of this structural feature. In MAP2c, such tertiary interactions are absent, which points to the importance of local structural constraints. In fact, urea and temperature dependence of the CD spectrum of its PRR reveals the presence of the extended and rather stiff polyproline II helix conformation that keeps the interaction site exposed. These data suggest that functionally significant residual structure exists in both of these IUPs. This structure, manifest as either transient local and/or global organization, ensures the spatial exposure of short contact segments on the surface. Pertinent data from other IUPs suggest that the presence of such recognition motifs may be a general feature of disordered proteins. To emphasize the possible importance of this structural trait, we propose that these motifs be called primary contact sites in IUPs

Interfacial Water at Protein Surfaces: Wide-Line NMR and DSC Characterization of Hydration in Ubiquitin Solutions

Wide-line 1H-NMR and differential scanning calorimetry measurements were done in aqueous solutions and on lyophilized samples of human ubiquitin between −70°C and +45°C. The measured properties (size, thermal evolution, and wide-line NMR spectra) of the protein-water interfacial region are substantially different in the double-distilled and buffered-water solutions of ubiquitin. The characteristic transition in water mobility is identified as the melting of the nonfreezing/hydrate water. The amount of water in the low-temperature mobile fraction is 0.4 g/g protein for the pure water solution. The amount of mobile water is higher and its temperature dependence more pronounced for the buffered solution. The specific heat of the nonfreezing/hydrate water was evaluated using combined differential scanning calorimetry and NMR data. Considering the interfacial region as an independent phase, the values obtained are 5.0–5.8 J·g−1·K−1, and the magnitudes are higher than that of pure/bulk water (4.2 J·g−1·K−1). This unexpected discrepancy can only be resolved in principle by assuming that hydrate water is in tight H-bond coupling with the protein matrix. The specific heat for the system composed of the protein molecule and its hydration water is 2.3 J·g−1·K−1. It could be concluded that the protein ubiquitin and its hydrate layer behave as a highly interconnected single phase in a thermodynamic sense