PROTEIN SURFACE SIMILARITIES EVALUATION FOR FUNCTIONAL ANNOTATION STUDIES

One of the main targets of bioinformatics is to assign functions to proteins whose function is unknown relying on homologies identifications with proteins with known functions. Several approaches are currently available: the best choice depends on the evolutionary distance that separates the protein of interest from its homologous. Recently attention has been focused on molecular surfaces since they do not depend on the three-dimensional structure and allow similarities to be identified which other methods can\u2019t identify. Furthermore, molecular surfaces are the interface of interaction between molecules, and their geometrical and physical descriptions will lead to the comprehension of the molecular recognition process, since the geometrical component has a fundamental role in the early stage of complex formation. This particular aspect would have a major impact in the field of drug design and in the understanding of the side effects due to interactions between proteins. During this thesis a protocol for similarities identification on molecular surfaces has been developed and optimized. In this process, molecular surfaces are calculated according to Lee Richard\u2019s model, and then are represented through triangular meshes. Successively surfaces are transformed into a set of object oriented images using a computer vision approach. This type of representation has the advantage of being independent from the position of the objects represented, and thus similar surfaces can be described by similar images. The search for similarities is then performed by indentifying correspondences between pairs of similar images, by filtering matches relying on geometrical criteria and then by clustering correspondences in high similarity groups. These groups are then used to align surfaces in order to evaluate results both by visual inspection and through appropriate indexes. This process can be applied in the field of functional annotation, through the identification of similarities between surfaces of homologous proteins, and in study of interaction between proteins, through the identification of complementary areas between interacting proteins. The whole process of similarities detection depends on the configuration of 15 parameters that balance the time needed to perform calculation with the quality of results found. The problem of parameters estimation has been addressed using an implementation of genetic algorithm, which allowed representing different configuration parameters as a population in which individuals that are able to align surfaces satisfactory are rewarded with an high fitness score. The effectiveness of the algorithm was then improved by the introduction of neighbor heuristic which reduced the computational time required for correspondence clustering on surfaces. Particular interest was placed in results displaying and in the construction of indices that can quantify the quality of results. Regarding the visualization problem, a display system was implemented based on the Visualization ToolKit libraries in order to represent surfaces aligned as objects in three-dimensional space, enabling the user to interact with the scene represented by changing the point of view or enlarging details of the scene represented. Regarding the definition of useful indexes for results evaluation, two indexes had a fundamental role. The first one, called overlap index, measures the percentage of vertices of two surfaces that are closer than 1 A\ub0 after the alignment. This index in particular is useful for evaluating the surface similarity since similar aligned surfaces will have a large number of vertices closer than this distance. The second index, called RMSD, is important because it evaluates the Root Mean Square Deviation of alpha carbons of two aligned proteins in the case of a complementary search. This index allows evaluating how the aligned protein is distant from the correct position in the crystal complex. Concerning results evaluation, we have noticed that the consideration of electrostatic potential allows assigning good scores in case of strong geometrical similarity in context of functional annotations, thus facilitating the identification of homologous surfaces. This method has been validated both in the search of similarities and in the search of complementarities. Regarding the search of similarities, we tried to analyze a sample of 13 known proteins with a prosite domain in order to identify the presence of such domains on molecular surfaces. For doing this, we first reduced the number of structures present in the Protein Data Bank to a group of representative structures. Then we calculated the molecular surfaces for each representative protein and we created a dataset of patches corresponding to the prosite functional domain. The test was then performed trying to align the surface of the 13 known proteins to the patches dataset of functional domains. The results showed that in most cases we are able to properly align a functional domain to a protein surface with the same functional domain, and that these evidence was easily identifiable both by the parameters used for results evaluations, both by visually inspecting the results of the alignments. The method was then tested for complementary research, trying to reconstruct the protein-protein complex present in a well known dataset used to validate docking methods. In the case of searching for similarities it is important to describe surfaces in details in order to increase the accuracy, but high precision when searching for complementarity is counterproductive, since the interaction between proteins is not only determined by geometrical features but also involves the formation of favorable electrostatic interactions and rearrangements of side chains. Thus molecular surfaces were calculated using smoothed surfaces, where most details are lost but allowing to detect more easily interacting surfaces. Results showed that the algorithm is able to align complexes with comparable scores than the programs currently available; Considering this experimental design and that the method does not take into account the electrostatic potential, we can assume that the results obtained are particularly interesting since the proposed method provides a wider set of conformations than other algorithms, upon which we can extend the analysis in order to identify a better prediction. In conclusions the proposed system is able to identify similarities on molecular surfaces through the analysis of images of local description. The results show that the system implemented is effective in identifying similar surface areas in the context of functional annotation. In regards to the search for complementarities, the algorithm seems to have an interesting perspective, even though the best complex proposed is not always biologically correct. From this point of view, we have to do more analysis in order to improve the methods in protein interaction studies

The relationship between structure and function in natural surfactant proteins

Surfactant activity is a property more commonly associated with small molecules than biological macromolecules. However, the significant advantages of improved biodegradability and biocompatibility that could be presented by natural surfactant proteins has elevated interest in a group of only a few proteins where intrinsic surfactant activity appears to be the primary function. Two examples of this group, ranaspumin-2 (Rsn-2) from the foam nests of the tungara frog and latherin, the surface active protein component of horse sweat, appear to be different from other surfactant proteins in the form of their activity. However, the exact molecular basis of this activity is poorly understood. This thesis describes work to rationalise surface activity and related properties in these unusual proteins. The properties of Rsn-2 and latherin including surface activity, interaction with lipid membranes and behaviour in solution were investigated to provide further insight into the characteristics that distinguish the surfactant proteins from both conventional surfactants and other proteins. A second protein component of the foam nests, ranaspumin-1 (Rsn-1), of previously unknown function was also found to be highly surface active and is proposed to function in a similar manner to Rsn-2. A model whereby Rsn-2 functions via a clamshell-like opening was tested through a combination of specialised NMR techniques and site-directed mutagenesis. The results identified features associated with surfactant activity, all of which were consistent with the model. The potential of Rsn-2 as a recombinant fusion partner for the production of functional surfactants or foams was proven by construction of a fluorescent conjugate. Solution state NMR was used to determine the structure of latherin. Information on the dynamic processes taking place in the molecule were derived by analysis of NMR relaxation data. The structure revealed is a super roll fold, similar to a single domain of the BPI-like proteins. A model is proposed whereby latherin recognises the air-water interface via three dynamic, hydrophobic loops at one end of its long cylindrical structure and then unfolds to expose its hydrophobic core at the air-water interface

Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance

Proteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.Proteorhodopsin (PR) wurde ursprünglich aus nicht kultivierten γ-Proteobakterium isoliert und ist in großen Mengen in den Ozeanen enthalten. PR ist wie sein homolog Bakteriorhodopsin (BR) ein TypI Retinal Bindeprotein und die Sequenzen sind zu 26% identisch. Innerhalb der PR Familie haben die Mitglieder eine Sequenzhomologie zu ungefähr 78% und zeigen einen Unterschied von 40 nm im absorptions spektrum. Diese Eigenschaft bietet ein gutes Modelsystem um zu verstehen durch welchen Mechanismus das Absorptionsspektrum moduliert wird. PR ist ein photoaktive Protonenpumpe und es wird angenommen, dass die Richtung des Protonentransfers vom pH-wert abhängt, was auf eine Rolle als ein pH abhängiger Regulator hindeutet. Da PR sowohl in der Zelle in hoher Zahl, als auch in den Ozeanen in unterschiedlichen Tiefen weit verbreitet ist, wird angenommen, dass PR bei der Verwertung von Sonnenlicht, im Energiestoffwechsel und beim Kohlenstoffumsatz beteiligt ist. Im Gegensatz zu BR, welches bekannterweise 2D Kristalle bildet, ist etwas vergleichbares für PR bis heute nicht bekannt. Weder der Mechanismus von PR noch seine 3D Struktur sind bisher gelöst. Die vorliegende Doktorarbeit versucht offene Punkte zum Mechanismus und zur Struktur von PR zu klären. Für die Charakterisierung werden 2D Kristallographie, "Atomic Force Microscopy" und Festkörper NMR verwendet. Für die Bildung von 2D Kristallen konnte eine große Auswahl an Kristallisationbedingungen ermittelt werden, was auf deutliche Protein Protein Wechselwirkungen hindeutet. Zieht man die hohe Zahl an PR Molekülen pro zelle in betracht, ist es wahrscheinlich, dass durch diese Interaktionen auch in der natürlichen Membran eine dichte Packung der Proteine auftritt. Elektronenmikroskopische Aufnahmen mit geringer Auflösung deuten auf eine ringförmige Anordnung der Proteine in einem hexagonalen Gitter mit einer Einheitszelle von 87Å * 87Å. Vorläufige FTIR Messungen deuten darauf hin, dass diese Anordnung den Photozyklus nicht behindert und sowohl K als auch M Zustand konnten identifiziert werden. Um weitere Informationen über den Oligomerisierungszustand der 2D Kristalle zu gewinnen wurden Einzelmolekül - und Rasterkraft Mikroskopie durchgeführt. Hierbei zeigte sich, dass das Protein in kristallinen und dicht rekonstituierten Regionen überwiegend als Hexamer vorliegt. Daneben kann zu einem geringen Anteil auch ein pentamerer Zustand beobachtet werden. Der Mechanismus der Proteinentfaltung war vergleichbar zu anderen, besser untersuchten Mitgliedern der Rhodopsinfamilie. Zwischen den Daten aus der "Atomic Force Microscopy" und der Elektronenmikroskopie zeigt sich eine gute Korrelation. Festkörper NMR an vollständig und selektiv markierten 2D Kristallen ergaben Spektren mit einer typischen 15N Linienbreite von 0,6 bis 1,2 ppm. Die 15N chemische Verschiebung der Schiffschen Base hat im Kristall den gleichen Wert wie funktional aktiv rekonstitutierte Proben, was indirekt die Funktionalität und die korrekte Faltung bestätigt. Die Zuordnung der 15N Signale für Tryptophan wurde durch "Rotational Echo Double Resonance" Experimente vorgenommen. 2D kreuzpolarisation Lee Goldburg Messungen zeigen den dynamischen Zustand des Proteins trotz der eingeschränkten Mobilität im kristallinen Zustand. Das Verhalten der Lipide wurde mit 31P messungen der Lipidkopfgruppe untersucht und zeigt, dass diese nicht fest gebunden sind, sondern sich mehr wie in einer Lipiddoppelschicht verhalten. Für 13C-13C homonukleare korrelations Experimente wurde die Mischzeit durch die Analyse von Aufbaukurven optimiert. Diese Versuche deuten darauf hin, dass es möglich ist einzelne Resonanzen aufzulösen, wie im Fall des Glutamat gezeigt mit einem gutem Signal zu Rauschen Verhältnis. Selektives "unlabeling" ist eine effizente Methode um die Ueberlappung der Signal zu reduzieren. Darüberhinaus sind für eine weitere Chrakterisisierung effizentere Markierungsschemata notwendig. Die bisherige spektrale Auflösung ist gut genug für die Untersuchung einzelner Aminosäuren, für vollständig markierte Proben sind weitere Verbesserungen notwendig

NMR studies of transiente protein complexes

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica,especialidade Bioquímica Física,pela Universidade Nova de Lisboa,Faculdade de Ciências e TecnologiaFundação para a Ciência e Tecnologia - Bolsa de Doutoramento (SFRH/BD/25342/2005)no âmbito do Programa Operacional Potencial Humano, da União Europeia (Fundo Social Europeu