PHYSICAL BIOCHEMISTRY

Author: DÍAZ MORENO IRENE.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: CENTRO DE INVESTIGACIONES CIENTIFICAS ISLA DE LA CARTUJA.
Place of preparation: INSTITUTO DE BIOQUÍMICA VEGETAL Y FOTOSÍNTESIS . UNIVERSIDAD DE SEVILLA CSIC..

Summary: Interactions trasitorias between biomolecules have become the center of the investigation into many areas of Biochemistry Moderna.La continued structural characterization using any of the structural high-resolution techniques, it is vital to understand the role of this type of these complexes. The work of this Doctoral Thesis has been raised with a double objetivo.Por one hand, in-depth knowledge of the molecular interactions and the structural characterization of the complex transition between proteins, targeting end compression factors that control the relationship between the structure and function of proteins, as well as ways of interaction and formation of this type of complex unstable or weak. has been given special emphasis on those features which give a protein their ability to interaccionear specifies transient with other . The second goal is an advance in the development and knowledge of two espectroscopias now essential for the study of interactions between biomolecules in solution: Nuclear Magnetic Resonance MRI and the absorption of X XAS.Este development Ray in turn has two different aspects: at trial, optimizing the quality of NMR spectra and XAS in concentrations límite.a level of methodology applied, so groundbreaking, XAS to study complex between biomolecules and addressing for the first time the carazterización structural between soluble proteins and membrane by NMR in solution.

Author: PERÁLVAREZ MARÍN ALEJANDRO.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE MEDICINA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The bacteriorodopsina (BR) is the simplest photosynthetic system is known, because it creates an electrochemical gradient from the energy of light, carrying protons across the membrane that is used by the ATP-sintasa for energy, in terms of lack of oxygen. Using methods for Molecular Biology mutagenesis protein, combined with biophysical studies, it is determined as timely changes in the protein sequence affect the structure and function of the protein. In this study we proposed to determine the role of prolinas included in the alfa-hélices B (Pro50), C (Pro91) and F (Pro186), in addition to the forces and interactions responsible for the situation and how distorted the propeller C the BR rise to the occasion Thr90-Pro91. This helix is the key mechanism of proton transport, as it contains the two waste espártico (Asp85 and Asp96) directly involved in transporting the proton. The biophysical studies conducted suggest that Prolinas included in the alfa-hélices transmembrane mainly play a dynamic role in Bacteriorodospsina. Regarding the reason Thr90-Pro91 in Hélice C stress the importance of this mechanism to develop a proper function of transporting protrón. The results for Bacteriorodopsina are extensible to other transmembrane proteins.

Author: LIDON MOYA MARIA DEL CARMEN.
Year: 2005.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR.
Place of preparation: INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR.

Summary: The main objective of this study was the identification and characterization of a compound with the ability to heterodimerizar with CA-C (carboxyl terminal domain of the protein from cápsida HIV-1), and thus prevent the assembly of the natural cápsida, and consequently, the infectivity of HIV / AIDS. To that end, the work is divided into three chapters. The first chapter has conducted thermodynamic characterization of CA-C using various biophysical techniques: fluorescence, DC, FTIR, NMR, and by size exclusion chromatography molecular different conditions of pH and temperature, in order to describe the stability of the monomer and dimérica forms of CA-C, and characterize any possible intermediary who could appear in the reaction of plegamiento-asociación of protein and energy to understand the factors that govern dimerización. The results indicate that the balance of thermal denaturation of CA-C consists of three reversible and independent thermal transitions: (i) the first transition (Tm 307 K), which is observed by NMR and FTIR, is the dissociation of dímero CA - C, leading to an intermediary monomer folded, (ii) a second transition (Tm 325 K), observed by fluorescence SLA, anisotropy and DC in the near ultraviolet, involves desplegamiento partially folded intermediate monomer, and (iii ) The third transition (Tm 332 K), which is observed by DC in the far ultraviolet, absorbance, NMR and FTIR carries the desplegamiento global broker monomer partially folded. In addition, we have also identified the thermodynamic parameters of decoupling and denatured forms dimérica and monomer of CA-Ca physiologic pH. It has been observed that CA-C, either monomer as dimérica, undergoes a transition conformacional at pH acid, forming a molten globule, whose properties are similar to those introduced intermediary monomer partially folded observed in the balance of thermal denaturation CA - C. The second chapter has carried out the design of a peptide (CAC1), which includes the sequence of the single propeller that forms the interface dimerización of CA-C (-hélice 2), and have studied its structure and properties partnership CA-C through various biophysical techniques: fluorescence, DC, NMR, by size exclusion chromatography molecular affinity chromatography analytical, and ITC. Experiments DC, by size exclusion chromatography molecular and NMR showed that CAC1 is able to interact with CA-C. Moreover, by fluorescence, affinity chromatography analytical, and ITC, it has been determined the apparent dissociation constant of heterocomplejo formed between CAC1 and CA-C, which is about 50 million, only five times higher than the entire domain (10 M). These results suggest that this peptide can be used as a model for designing an antiviral agent. And the third chapter has undertaken the design of protein-based structure CA-C, but smaller, which also contains all the -hélice 2, also contain other residues critical for dimerización. These miniproteínas were generated through protein engineering techniques, and has tried to carry out his expression and purification. None of the changes designed to obtain miniproteínas that contain only until the second propeller of CA-C allowed the expression of protein, but there has been the expression and tuning protocol purification of a fragment CA-C it contains residues 146-214 domain intact, for the subsequent conduct of biophysical studies.

Author: LEÓN MADRENAS XAVIER.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE MEDICINA.
Place of preparation: UNITAT DE BIOFÍSICA.

Author: SOT SANZ JESÚS.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: In order to better understand the phenomenon of rafts and the effects of ceramides in the cell membrane, we conducted a series of studies in biophysical model vesicles. At first we studied the effect of lipid composition in resistance to the solubilization by Triton X-100 leading cause of this resistance. To this end analyzed the effect of Triton X-100 in vesicles formed mainly by PC / SM / Ch. It was observed that must be present esfingomielina (and not others esfingolípidos) and cholesterol to cause resistance to the solubilization by Triton X-100 and that this resistance was higher than 37Â ° C to 4Â ° C, decreasing as increasing the proportion PC. This behavior was not only dependent on the existence of an orderly phase liquid, as in similar proportions of lipid liquid sorted, the interaction between the group of cholesterol and hydroxy group arbonilo of the SM, increases resistance wing by solubilization Triton X-100. Under conditions in which there was a partial solubilization, waste is not solubilized had a composition close to PC / SM / Ch (1:1:1), which can not be sure that membrane resistant to detergents correspond to existing structures in the membrane or are the result of a partial solubilization and subsequent reesamblaje of bicapas lipid. Continuing experiments solubilization were looking for a simple lipid mixture should behave as the rafts, or whether they were resistant to solubilization at low temperatures, becoming soluble with increasing temperature, this was achieved with the sample SM / Be . Looking at the composition of waste resistant to solubilization was observed that are enriched in Cer, in addition to happen this resistance was necessary packaging a high lipid hydrocarbon chains, so this resistance only occurred with cermidas long chain and with saturated phospholipids. Using DSC and fluorescence decay, it was observed that the existence of a small amount of Cer produced the appearance of ordered domains that increased with the proportion of ceramide also established that the merger of these domains attached to the increase in solubilization. Finally, we studied the process of solubilization in GUVs of SM / Cer by fluorescence microscopy observed that in the process of solubilisation the Triton X-100 acting on a preferential basis on the SM-rich domains without affecting so apparent domains rich ceramide. The last thing we studied were the effects that produced the ceramides long chain (Cer16) and short (Cer6 and CEr2) in the lipid membranes. Through studies of topcoat, it was observed that both the Cer2 as Cer6, behaved as anfifilos soluble, so that they were able to solubilizar topcoat and bicapas, but could not get solubilizar completely bilayer. Through other techniques (DSC, 31P-RMN, and X-ray diffraction), we studied the effect that produced these ceramides in the transition gel-fluido and lamelar-hexagonal of phospholipids. The results showed that there were two types of interaction ceramida-fosfolípido, corresponding to the ceramides long chain or short. Representatives can be considered characteristic of these two classes the Cer16 and Cer2 respectively. The Cer16 mixed little with phospholipids in the gel phase, although solubilizaba phase fluid. Furthermore, the transition lamelar-hexagonal. By contrast, the Cer2 mixed well with phospholipids in the gel phase, stabilization phase fluid. The Cer6 show intermediate properties.

Author: CONTRERAS GÓMEZ FRANCESC-XABIER.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].

Summary: This thesis has been studying the changes induced both by the activity esfingomielinasa as the ceramides, the structure and physical properties of the cell membrane. To this end, it has been observed how cholesterol allows the degradation of esfingomielinasa even at temperatures below the temperature of transition gel-fluido of esfingolípido due to the formation of phases líquido-ordenadas. In addition, it has been observed how ceramides, already generated in situ by the esfingomielinasa, or already added externally induced movement trasnbicapa (flip-flop) lipids in biological membranes and membrane model. Finally, it describes how the ceramides inhibit the activity scramblasa of the membranes by a mechanism independent of PKC, and located with the scramblasa in apoptotic cells.

Author: LORIZATE NOGALES MAIER.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: UNIVERSIDAD DEL PAIS VASCO.
Place of preparation: UNIDAD DE BIOFÍSICA (CENTRO MIXTO CSIC-UPV).

Summary: This thesis has deepened in the study of the regions of ectodominio gp41 of HIV-1 that have the capacity to interact with membranes. Specifically is the recognition by antibodies used as a criterion for establishing the states involved in membrane or physiologically relevant solution. The sequence domain interface (preTM), which is adjacent to membrane is stabilized as oligoméricos at different stages of the protein in the virión. In addition, the epítopo recognized by the antibody neutralizing 4E10 which is in the sequence, recognize the epítopo immersed in membrane type rafts. Finally, it has been determined that at the stage of the native protein gp41, and the fusion peptide sequence nfipática and adjacent to the domain interface (preTM), USAID, interact, and that this interaction would provide some of this complex solubility, thus avoiding in the merger process catalyzed by gp41, sequences that have much affinity for membranes not interact with it before being activated process. Furthermore, this interaction would create a structure not leicoidal in the region AID structure recognized by the antibody neutralizing 2F5.