RESUMO
More than 60 years of biochemical and biophysical studies have accustomed us to think of proteins as highly purified entities that act in isolation, more or less freely diffusing until they find their cognate partner to bind to. While in vitro experiments that reproduce these conditions largely remain the only way to investigate the intrinsic properties of molecules, this approach ignores an important factor: in their natural milieu , proteins are surrounded by several other molecules of different chemical nature, and this crowded environment can considerably modify their behaviour. About 40% of the cellular volume on average is occupied by all sorts of molecules. Furthermore, biological macromolecules live and operate in an extremely structured and complex environment within the cell (endoplasmic reticulum, Golgi apparatus, cytoskeletal structures, etc). Hence, to further complicate the picture, the interior of the cell is by no means a simply crowded medium, rather, a most crowded and confining one. In recent times, several approaches have been developed in the attempt to take into account important factors such as the ones mentioned above, at both theoretical and experimental levels, so that this field of research is now emerging as one of the most thriving in molecular and cell biology (see figure 1). [Formula: see text] Figure 1. Left: number of articles containing the word 'crowding' as a keyword limited to the biological and chemical science domains (source: ISI Web of Science). The arrow flags the 2003 'EMBO Workshop on Biological Implications of Macromolecular Crowding' (Embo, 2012). Right: number of citations to articles containing the word 'crowding' limited to the same domains (bars) and an exponential regression curve (source: Elsevier Scopus). To promote the importance of molecular crowding and confinement and provide researchers active in this field an interdisciplinary forum for meeting and exchanging ideas, we recently organized an international conference held in Ascona from 10 to 14 June 2012. In the unique scenario of the Maggiore lake and absorbed in the magic atmosphere of the Centro Stefano Franscini (CSF) at Monte Verità, we enjoyed three-and-a-half days of intense and inspiring activity, where not only many of the most prominent scientists working on macromolecular crowding, but also experts in closely related fields such as colloids and soft matter presented their work. The meeting was intended and has been organized to bring theoreticians and experimentalists together in the attempt to promote an active dialogue. Moreover, we wanted different disciplines to be represented, notably physics and chemistry, besides biology, as cross-fertilization is proving an increasingly fundamental source of inspiration and advancement. This issue of Physical Biology (PB) features a selection of the oral contributions presented at the conference, expanded in the form of research or review articles. PB, one of the scientific journals of the Institute of Physics (IOP), is one of the most dynamic and lively forums active at the interface between biology on one side, and physics and mathematics on the other. As its mission is stated by IOP, PB 'focuses on research in which physics-based approaches lead to new insights into biological systems at all scales of space and time, and all levels of complexity'. For these reasons, and also in view of its high reputation and broad readership, PB appears to be the ideal place for disseminating the thriving pieces of research presented at the conference. We are extremely grateful to PB and its kind and efficient editorial staff who helped make this issue a great scientific follow-up to the conference. The opening lecture of the conference, the first of four day-opening keynote lectures, was given by Allen P Minton from NIH (USA), possibly the most influential among the pioneers in the field. He provided a lucid and well-thought-out overview of the concept of macromolecular crowding through an exhaustive chronological account of the major milestones. It is clear that the concept of excluded volume as a key factor remains central to the concept of molecular crowding. As a consequence, simple descriptive paradigms borrowed essentially from colloid physics may still provide useful tools to understand the subtle effects of crowding and confinement in living matter. The contiguity between crowding, colloids and soft matter further emerged as an important concept in the course of the conference in several theoretical lectures and a few experimental ones. Dave Thirumalai, from the University of Maryland (USA), one of the most active theoreticians in the field of theoretical biophysics, outlined scaling theories, concepts from colloid literature and different simulation techniques to describe scenarios for crowding-induced changes in the structure and dynamics of proteins and RNA. In particular, he showed the importance of the shape of crowding particles in affecting folding oligomerization of amyloidogenic peptides. Johannes Schöneberg, from IMPRS, Mathematics Institute (Germany), illustrated ReaDDy , a newly developed particle-based simulation software tool for reaction-diffusion dynamics, developed in the group of Frank Noe at EMPRS. He showed that ReaDDy makes it possible to bridge the gap between soft matter and molecular dynamics (MD) simulations on the one hand and particle-based stochastic reaction-diffusion simulations on the other. We asked Johannes to organize a tutorial session to lead interested participants into the package and 'get their hands wet' under the guidance of the developers. The tutorial session was indeed successful and the broad possibilities offered by the simulation toolkit appeared to be clear to the participants. Paolo De Los Rios, from the Ecole Polytechnique Fédérale de Lausanne (EPFL, Switzerland), examined the complexity of the effects caused by crowding conditions from the point of view of statistical physics. Starting from a modification of the well-known Smoluchowski approach to calculate the encounter rate of diffusion-limited reactions, he showed how more realistic situations accounting for crowding effects could be treated equally well on the same theoretical grounds. This talk marked an important point in the conference as it reinforced the idea that simple models of theoretical physics still have the power to provide inspiring results in spite of the intrinsic simplifications of such theoretical approaches. Along the same lines, Nicolas Dorsaz, from the University of Cambridge (UK), proposed an extension of the Smoluchowski framework that incorporates repulsive and attracting interactions between the reactants. This approach was illustrated by reaction rates obtained from event-driven Brownian dynamics and dynamical Monte Carlo simulations. Another striking example of the physical subtleties associated with modelling crowding effects was provided by Jeffrey Skolnick, from the Georgia Institute of Technology (USA). He examined the role of hydrodynamic interactions in the self-organization of biological assemblies in the presence of crowding. His results strongly suggest that hydrodynamic interactions greatly affect the kinetics of self-assembly reactions, so that including them in the picture appears crucial for understanding the dynamics of biological systems in vivo . Margareth Cheung, from the University of Houston (USA), emphasized that how the crowded environment inside a cell affects the structural conformation of a protein with a spherical shape is a vital question because the geometry of proteins and protein-protein complexes are far from globules in vivo . Her work demonstrates the malleability of 'native' proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo . Huan-Xiang Zhou, from the Florida State University (USA), focused on atomistic simulations of protein folding and binding under crowding conditions. His lab has developed a post-processing method that allows the atomistic representation of proteins in folding and binding processes under crowding. A comparison with experimental results was also presented. Other lecturers pointed out that there are still aspects not entirely explored in the effects of both crowding and confinement. As suggested in the talk by Gary Pielak, from the University of North Carolina (USA), the currently used synthetic crowding agents are far from being satisfactory in replicating naturally occurring effects associated with crowded environments. For example, non-specific binding seems to play a subtle role in the cell, as natural macromolecules can induce both stabilization and destabilization when used as crowders. It is indeed possible to fine-tune the effect of proteins, as crowders, on the stability of other proteins. Another aspect that became clear is that new, more powerful methods need to be developed to study the effect of crowding, but even more to compare crowding and confinement. Indeed, it appeared clear from the lecture by Pierandrea Temussi, from the University of Naples (Italy), that a reliable comparison of the effects of crowding and confinement on the stability of proteins can only be based on the measurement of the whole stability curve of the same protein. Controversial aspects do not pertain only to the influence of crowding on protein stability, but also to aggregation phenomena in natural fluids. Domenico Sanfelice, from NIMR (London, UK), reported an interesting case of the apparent influence of crowding on aggregation. Hen egg white, a possible natural medium to study macromolecules in crowded conditions can dramatically increase the aggregation kinetics of proteins with an inbuilt tendency to associate. By carefully dissecting the phenomenology, it was shown that only part of this effect is due to crowding, while another factor playing an important role is the interaction withproteins from the milieu . In other words, high-molecular-weight glycoproteins can act as efficient molecular seeds for aggregation. A special topic of great relevance in the conference appeared to be the direct study of crowding in living systems. Alan Verkman, from the University of California, San Francisco (USA), one of the world's leading scientific personalities in the field of experimental investigation of crowding and confinement, was invited to give the second plenary lecture devoted to the experimental study of crowding effects in vivo . In his keynote lecture, Dr Verkman led us on a wide and compelling tour, exploring the main experimental approaches to study molecular crowding in and around cells. After a thorough examination of methods such as fluorescence recovery after photo-bleaching, fluorescence correlation spectroscopy, photo-activation localization microscopy and stochastic reconstruction microscopy, he concluded that the general consensus emerging from experimental studies is that the notion of universally anomalous diffusion in and around cells as a consequence of molecular crowding may not be correct, and that the slowing of diffusion in cells is less marked than has been widely assumed and can be simply described through a five- to sixfold reduction of the normal diffusion coefficient. A Soranno, from the University of Zürich (Switzerland), described how, by employing FRET measurements, it is possible to quantify the effect of molecular crowding on the dimensions of the highly charged, intrinsically disordered protein human prothymosin alpha. For a large variety of polymeric crowders (PEG, PVP, Ficoll, Dextran, PVA, PAA), a collapse of the polypeptide chain is observed with increasing polymer size and polymer concentration. The largest extent of collapse is observed for polymer radii comparable to the dimensions of the protein, in agreement with theoretical considerations. For his contribution, A Soranno was awarded the CSF Award for the best contributed talk. In his most inspiring talk, Clifford Brangwynne, from Princeton University (USA), drew attention to very important objects, namely Ribonucleoprotein (RNP) bodies. These are non-membrane-bound macromolecular assemblies that form from the dynamic interactions of RNA and proteins. The assembly of RNP bodies may sensitively depend on the biophysical features of the surrounding cytoplasm, including the degree of crowding, transport coefficients and mechanical properties. This dependency may have important implications for the RNA processing reactions involved in fundamental biological processes such as developmental cell growth. Remarkably, Brangwynne showed how RNPs behave in the cell as liquid droplets, pointing to a possible entirely new means that the cell could use to control and fine-tune its internal processes, in fact, more than that, a completely unexplored, new state of organization of living matter, and a functional one. Giuseppe Zaccai, from Institut Laue Langevin, Grenoble (France), showed that protein dynamics is more sensitive than structure to environmental factors such as crowding, solvent, temperature or pressure. Furthermore, he convincingly explained how neutron scattering provides unique experimental data to underpin MD calculations in this context. Following up on environment-induced modulations of protein functional dynamics, Ruth Nussinov, from Tel Aviv University (Israel), addressed the important problem of whether cellular signals can travel long distances in a crowded environment. She proposed a model based on the evolution of at least three properties: a modular functional organization of the cellular network, sequences in some key regions of proteins, such as linkers or loops, and compact interactions between proteins, possibly favoured by a crowded environment. The workshop ended on a keynote lecture by Jean-Marie Lehn, from the Université de Strasbourg (France). Lehn, 1987 Nobel Laureate in chemistry, offered a 'supramolecular view' of the field of molecular interactions. Supramolecular chemistry explores the design of systems undergoing self-organization , i.e. systems capable of generating well-defined functional supramolecular architectures by self-assembling from their components, thus behaving as programmed chemical systems . Chemistry may therefore be considered an information science , the science of informed matter. Supramolecular chemistry is intrinsically a dynamic chemistry in view of the ability of the interactions connecting the molecular components of a supramolecular entity and the resulting ability of supramolecular species to exchange their constituents. The same holds for molecular chemistry when the molecular entity contains covalent bonds that may form and break reversibly, so as to allow a continuous change in constitution by the reorganization and exchange of building blocks. These features define a constitutional dynamic chemistry (CDC) on both the molecular and supramolecular levels. CDC takes advantage of dynamic constitutional diversity to allow variation and selection in response to either internal or external factors to achieve adaptation . The merging of the features-information and programmability, dynamics and reversibility, constitution and structural diversity-points towards the emergence of adaptive and evolutive chemistry . The whole workshop could have not taken place without the help of the Centro Stefano Franscini. The CSF is the congress centre of the Swiss Federal Institute of Technology of Zurich (ETH Zurich) and has been situated at Monte Verità since 1989. It is an ideal meeting point for all members of the international scientific community who wish to discuss the state-of-the-art and new challenges of any field of research. The CSF supports 20-25 international conferences every year and, since 2010, up to ten winter doctoral schools1. The competence and professionalism of the staff were at the same level of beauty and inspiring character as that of Monte Verità. A meeting of this sort, if successful, leaves the audience with more open questions than settled answers, and this was definitely the case for Crowding 2012. Excluded volume is clearly a fundamental concept that has allowed crowding, a very familiar concept in soft matter, to enter into the domain of biological sciences. However, the complexity of the biological milieu calls for more refined descriptions. What is the role of electrostatic and electrodynamic interactions? What is the role of hydrodynamics interactions? To what extent does the strong spatial inhomogeneity (clustering of molecules, cellular compartmentalization, etc) have to be taken into account? Or, more generally, what are the minimal elements that prove crucial to describe reactions within a cell? How does the diffusion proceed (diffusion, slow diffusion, sub-diffusion) given that the experimental evidences are still controversial? In conclusion, we knew that allowing scientists with very different backgrounds and ideas to mingle was a hazardous attempt. Despite that, the workshop turned out to be a very successful experiment, which was highly enjoyed both by the participants and the organizers. Discussions sparked regularly among ever-changing groups, comprising senior scientists and students, despite the rather tight schedule, adding to the sense of fulfilment ignited by the outstanding level of the presentations. Given the success of the meeting Crowding 2012, a new event has been organized and will take place on the same themes during fall 2013, this time in the beautiful scenery of the Loire valley in France. The workshop 'Macromolecular crowding effects in cell biology: models and experiments' will be held on the CNRS campus in Orléans, France, on 24-25 October 2013. More information can be found on the workshop website: http://dirac.cnrs-orleans.fr/â¼piazza/. 1Source: www.csf.ethz.ch/
RESUMO
In living systems, proteins are surrounded by many other macromolecules of different nature, at high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, such as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides or even synthetic polymers. We have recently proposed hen egg white (HEW) as a suitable, natural medium to study macromolecules in crowding conditions. Here, we show that HEW can increase dramatically the aggregation kinetics of proteins with an in-built tendency to associate. By dissecting the mechanism we demonstrate that only part of this effect is due to crowding, while another factor playing an important role is the interaction with proteins from the milieu. High molecular weight glycoproteins present in HEW act as efficient molecular seeds for aggregation. Our results bear important consequences for in-cell NMR studies and suggest a role of glycosylated proteins in aggregation.
Assuntos
Galinhas , Proteínas do Ovo/química , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Animais , Proteínas do Ovo/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Cinética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
A few proteins, discovered mainly in tropical fruits, have a distinct sweet taste. These proteins have played an important role towards a molecular understanding of the mechanisms of taste. Owing to the huge difference in size, between most sweeteners and sweet proteins, it was believed that they must interact with a different receptor from that of small molecular weight sweeteners. Recent modelling studies have shown that the single sweet taste receptor has multiple active sites and that the mechanism of interaction of sweet proteins is intrinsically different from that of small sweeteners. Small molecular weight sweeteners occupy small receptor cavities inside two subdomains of the receptor, whereas sweet proteins can interact with the sweet receptor according to a mechanism called the 'wedge model' in which they bind to a large external cavity. This review describes these mechanisms and outlines a history of sweet proteins.
Assuntos
Proteínas de Plantas/química , Edulcorantes/química , Paladar , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Endogenous opioids have been studied extensively since their discovery, in the hope of finding a perfect analgesic, devoid of the secondary effects of alkaloid opioids. However, the design of selective opioid agonists has proved very difficult. First, structural studies of peptides in general are hampered by their intrinsic flexibility. Second, the relationship between constitution and the so-called 'bioactive conformation' is far from obvious. Ideally, a direct structural study of the complex between a peptide and its receptor should answer both questions, but such a study is not possible, because opioid receptors are large membrane proteins, difficult to study by standard structural techniques. Thus, conformational studies of opioid peptides are still important for drug design and also for indirect receptor mapping. This review deals with conformational studies of natural opioid peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yields a convincing bioactive conformation, but the global conformation of longer peptides in biomimetic environments can shed light on the interaction with receptors.
Assuntos
Peptídeos Opioides/química , Conformação Proteica , Animais , Sítios de Ligação , Humanos , Modelos Biológicos , Modelos Moleculares , Peptídeos Opioides/metabolismo , Peptídeos Opioides/farmacologia , Receptores Opioides/química , Receptores Opioides/metabolismo , Soluções , SolventesRESUMO
Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.
Assuntos
Aprotinina/química , Peptídeos/química , Espectroscopia de Ressonância MagnéticaRESUMO
The change of selectivity and the induction of antagonism by the insertion of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the second position of several opioid peptides have led to the interpretation of Tyr-Tic as a specific message domain for delta-opioid antagonists and to the discovery of dipeptides with substantial opioid activity. Selectivity and activity increase enormously when Tyr is substituted by 2',6'-dimethyl tyrosine (Dmt), hinting that the side chain of Dmt fits a hydrophobic cavity of the receptor very tightly and precisely. We have investigated the specificity of this fit by systematic changes of the substituents on the aromatic ring of ryr. Mono- and disubstitutions different from 2',6'- invariably lead to catastrophic decreases of activity. The only substitution compatible with retention of substantial antagonism is 2-methyl. An analysis of the conformational properties of all analogues reveals that substitutions do not affect the global shape of the molecule significantly. Accordingly, it is possible to use the shape of the different side chains to map the hydrophobic cavity of the receptor. The resulting complementary image is funnel shaped.
Assuntos
Encéfalo/metabolismo , Dipeptídeos/metabolismo , Isoquinolinas/química , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/metabolismo , Tetra-Hidroisoquinolinas , Tirosina/análogos & derivados , Tirosina/metabolismo , Substituição de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Conformação Molecular , Naltrexona/metabolismo , Antagonistas de Entorpecentes/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/antagonistas & inibidores , Termodinâmica , Tirosina/químicaRESUMO
The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Plantas/química , Edulcorantes/química , Modelos Moleculares , Proteínas de Plantas/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Água/químicaRESUMO
Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.
Assuntos
Fármacos Anti-HIV/química , Endorribonucleases/química , Epitopos/química , Oligopeptídeos/síntese química , Peptídeo T/química , Ribonuclease Pancreático/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/fisiologia , Antígenos CD4/química , Antígenos CD4/metabolismo , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Desenho de Fármacos , Estabilidade de Medicamentos , Endorribonucleases/farmacologia , HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Conformação Molecular , Mimetismo Molecular/fisiologia , Monócitos/citologia , Monócitos/metabolismo , Oligopeptídeos/farmacologia , Peptídeo T/análogos & derivados , Peptídeo T/farmacologia , Ribonuclease Pancreático/farmacologiaRESUMO
Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90 degrees C, the Cd-thiolate chromophore absorbance at 254 nm of mouse MT was not modified up to 60 degrees C, whereas the absorbance of fish MT decreased significantly starting from 30 degrees C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260 nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.
Assuntos
Metalotioneína/química , Perciformes/metabolismo , Sequência de Aminoácidos , Animais , Cádmio/metabolismo , Dicroísmo Circular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Cinética , Espectroscopia de Ressonância Magnética , Metalotioneína/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resorcinóis/metabolismo , Alinhamento de Sequência , Espectrofotometria Atômica , Temperatura , Zinco/metabolismoRESUMO
The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha-helix cradled into the concave side of a five-strand anti-parallel beta-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main beta-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 microM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Engenharia de Proteínas , Edulcorantes/química , Sequência de Aminoácidos , Cistatinas/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Soluções , Propriedades de Superfície , TemperaturaRESUMO
Orphanin FQ2 (OFQ2) is a novel heptadecapeptide generated from prepronociceptin (PPNOC), the same precursor of nociceptin/orphanin FQ and nocistatin. OFQ2 is a potent analgesic when administered both supraspinally and spinally. In order to clarify the structural relationship with all peptides generated from PPNOC, we have undertaken the conformational study of OFQ2 in water and in structure-promoting solvent media. Nuclear magnetic resonance data and theoretical calculations are consistent with a well defined helical structure from Met(5) to Ser(16). The uniform distribution of hydrophobic residues along the helix suggests that OFQ2 may interact with the transmembrane helices of a receptor akin to those of nociceptin and opioids.
Assuntos
Analgésicos Opioides/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , SoluçõesRESUMO
Nocistatin, a new heptadecapeptide encoded in the bPNP-3 gene, has a powerful biological activity connected with the mechanisms of pain transmission. It does not bind to the opioid receptors but to another brain receptor with high affinity. In order to substantiate these novel biological data with a structural basis, we have undertaken a conformational study in solution. Proton nmr data in helicogenic solvents are consistent with a well-defined helical structure that is consistent with the nmr parameters of the C-terminal octapeptide, a shorter fragment that retains allodynia-blocking activity.
Assuntos
Analgésicos Opioides/química , Peptídeos Opioides/química , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Ratos , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
TEMPOL, the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, has been used to determine the surface characteristics of tendamistat, a small protein with a well-characterised structure both in solution and in the crystal. A good correlation has been found between predicted regions of exposed protein surface and the intensity attenuations induced by the probe on 2D NMR TOCSY cross peaks of tendamistat in the paramagnetic water solution. All the high paramagnetic effects have been interpreted in terms of more efficient competition of TEMPOL with water molecules at some surface positions. The active site of tendamistat coincides with the largest surface patch accessible to the probe. A strong hydration of protein N and C termini can also be suggested by this structural approach, as these locations exhibit reduced paramagnetic perturbations. Provided that the solution structure is known, the use of this paramagnetic probe seems to be well suited to delineate the dynamic behaviour of the protein surface and, more generally, to gain relevant information about the molecular presentation processes.
Assuntos
Óxidos N-Cíclicos/metabolismo , Peptídeos/química , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Prótons , Solventes/metabolismo , Marcadores de Spin , Temperatura , Água/metabolismo , alfa-Amilases/antagonistas & inibidoresRESUMO
Beta-endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.
Assuntos
beta-Endorfina/química , Sequência de Aminoácidos , Animais , Dimetil Sulfóxido , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Solventes , ÁguaRESUMO
Dynorphin A, the endogenous agonist for the kappa opioid receptor, has been studied by NMR spectroscopy in methanol, acetonitrile, DMSO and in mixtures of hexafluoroacetone/water and DMSO/water. NMR data in the DMSO/water cryomixture at 278 K are consistent with a conformer in which the N-terminal part, like the corresponding message domain of enkephalins, is poorly ordered, whereas the C-terminal part is folded in a loop centred around Pro10. The folded structure of the C-terminal part (address moiety) may shed light on the role of the essential residues Arg7, Lys11 and Lys13.
Assuntos
Dinorfinas/química , Sequência de Aminoácidos , Temperatura Baixa , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Receptores Opioides/química , Receptores Opioides kappa/química , Receptores Opioides mu/química , Soluções , Solventes , Receptor de NociceptinaRESUMO
Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-[T-K-P-R-G], has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-[T-K-P-R-G] based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-[T-K-P-R-G] and of the beta-turn conformation proposed by Suzuki for the [T,S]-P-K-R motif.
Assuntos
Antineoplásicos/química , Peptídeos Cíclicos/química , Tuftsina/química , Crioprotetores , Dimetil Sulfóxido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , ÁguaRESUMO
The most dramatic, but seldom mentioned, difference between alkaloid and peptide opioids is the change of chirality of the alpha carbon of the tyramine moiety. We propose that the presence of Gly2 or D-Ala2 in the two most common message domains compensates this change by allowing the attainment of unusual conformations. A thorough conformational search of Tyr-D-Ala-Phe-NH-CH3 and of its isomer Tyr-L-Ala-Phe-NH-CH3 backs this view and establishes a solid link between alkaloid and peptide opioids. This finding supports the notion that morphine, like other neurologically active plant compounds, may bind to endogenous receptors in plants to regulate cell-to-cell signaling systems.
Assuntos
Hormônios/química , Proteínas de Plantas/química , Aminoácidos/química , Modelos Moleculares , Oximorfona/análogos & derivados , Oximorfona/química , Conformação Proteica , Compostos de Espiro/químicaRESUMO
Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu-enkephalin was synthesized with 15N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSOd6/H2O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure-activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. beta- and gamma-turns) and finally to select only those consistent with the requirements of delta selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new delta selective opioid peptides.
Assuntos
Encefalina Leucina/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica , Temperatura Baixa , Encefalina Leucina/fisiologia , Modelos Moleculares , Isótopos de Nitrogênio , Software , Relação Estrutura-Atividade , Anormalidade TorcionalRESUMO
Substitution of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in place of Gly2 in dynorphin A-(1-13)-NH2 and -(1-11)-NH2 (DYN) analogues (1 and 2) decreased the affinity to the kappa, delta, and mu receptors, and kappa selectivity. The analogue [D-Ala2, des-Gly3]DYN (4), a chimera between deltorphin/dermorphin N-terminal tripeptide and DYN, was virtually inactive for kappa-sites while the affinities for delta- and mu-receptors remained essentially unchanged. The doubly substituted analogue [2',6'-dimethyl-L-tyrosine (Dmt1)-Tic2]DYN (3) exhibited high delta-affinity (Ki=0.39 nM) while mu- and kappa-affinities were only an order of magnitude less (4-5 nM). Bioactivity of [Tic2]DYN peptides (1-3) on guinea-pig ileum and rabbit jejunum revealed potent delta- and kappa-antagonism, while the delta agonist potency of 4 was comparable to DYN. Thus, conversion from a kappa-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with delta- and mu-opioid agonists containing a Tic2 residue.
Assuntos
Analgésicos Opioides/farmacologia , Dinorfinas/farmacologia , Músculo Liso/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Tetra-Hidroisoquinolinas , Analgésicos Opioides/síntese química , Animais , Relação Dose-Resposta a Droga , Desenho de Fármacos , Dinorfinas/síntese química , Eletrofisiologia , Cobaias , Íleo , Isoquinolinas/química , Contração Muscular/efeitos dos fármacos , Oligopeptídeos/química , Fragmentos de Peptídeos/síntese química , Coelhos , Relação Estrutura-AtividadeRESUMO
We have recently designed potent delta selective opioid antagonist dipeptides on the basis of a simple conformational analysis. Following a similar procedure we found a mu selective dipeptide antagonist, 2,6-dimethyl-Tyr-D-Phe-NH2. Although its selectivity is not as high as those of the quoted delta selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6-dimethyl-Tyr-D-Phe message, like the delta selective 2,6-dimethyl-Tyr-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid counterpart, seems able to impart antagonism to longer peptides.