Biomedical subjects

Alessandro Vergara

Publications and source records attributed to Alessandro Vergara.

7 recordsLinked to original sources

PubMed2006

High resolution crystal structure of deoxy hemoglobin from Trematomus bernacchii at different pH values: the role of histidine residues in modulating the strength of the root effect.

The Root effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen-binding ability at acidic pH. Here, we report the high-resolution structure of the deoxy form of Hb isolated from the Antarctic fish Trematomus bernacchii (HbTb) crystallized at pH 6.2 and 8.4. The structure at acidic pH has been previously determined at a moderate resolution (Ito et al., J Mol Biol 1995;250:648-658). Our results provide a clear picture of the events occurring upon the pH increase from 6.2 to 8.4, observed within a practically unchanged crystal environment. In particular, at pH 8.4, the interaspartic hydrogen bond at the alpha(1)beta(2) interface is partially broken, suggesting a pK(a) close to 8.4 for Asp95alpha. In addition, a detailed survey of the histidine modifications, caused by the change in pH, also indicates that at least three hot regions of the molecule are modified (Ebeta helix, Cbeta-tail, CDalpha corner) and can be considered to be involved at various levels in the release of the Root protons. Most importantly, at the CDalpha corner, the break of the salt bridge Asp48alpha-His55alpha allows us to describe a detailed mechanism that transmits the modification from the CDalpha corner far to the alpha heme. More generally, the results shed light on the role played by the histidine residues in modulating the strength of the Root effect and also support the emerging idea that the structural determinants, at least for a part of the Root effect, are specific of each Hb endowed with this property.

Amino Acid Motifs↗

PubMed2006

Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi.

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root-effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146beta and His69beta, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95alpha, Asp99beta, and Asp101beta should not be appropriate for the formation of Asp-Asp interactions, which are important for the Root effect. Here, we report the high-resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41alpha and Ser97alpha at the alpha1beta2 interface does not prevent the formation of the inter-Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95alpha and Asp101beta, which is stabilized by Asp99beta, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root-effect Hbs.

Animals↗

PubMed2005

Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology.

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.

Animals↗

PubMed2003

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 3. Analysis of residual acceleration-induced motion.

(PPG)(10) crystallization experiments onboard the ISS using the Advanced Protein Crystallization Facility have shown parallel and coherent crystal motions. The residual acceleration profiles and the History of the ISS Increment 3 mission allow a quantitative interpretation of these motions. Two events determine the observed crystal motions: the undocking of the Space Shuttle and a change in the ISS attitude required for power generation. No correlation between these motions and the crystal quality is apparent.

Collagen↗

PubMed2002

Physical aspects of protein crystal growth investigated with the Advanced Protein Crystallization Facility in reduced-gravity environments.

The physicochemical aspects of protein crystallization in reduced-gravity environments ( micro g) have been investigated with the Advanced Protein Crystallization Facility during six space missions. This review summarizes the results, dealing with the mechanisms of nucleation and crystal growth and with the quality of the crystals that were obtained under reduced gravity as well as under normal gravity on earth. Statistical analyses of the experimental data strongly support the fact that micro g has a positive effect on crystallization and on crystal quality. A comparison of experiments and theories of protein crystallization in reduced-gravity environments is presented. Recommendations for improving the performance of protein crystallization experiments in micro g and on earth are discussed.

Animals↗

PubMed2002

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 1. Video observation.

Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.

Collagen↗

PubMed2002

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 2. Comparison of crystal quality by X-ray diffraction.

Crystals of the collagen-like polypeptide (PPG)(10) were obtained within the Advanced Protein Crystallization Facility on board the International Space Station, during the STS-105/STS-108 mission. The duration of this mission was such to ensure that the crystallization process had reached its end. Crystals were grown both in the presence and in the absence of agarose gel, to compare the quality of the crystals obtained from these different environments. As a result, crystals grown in the absence of agarose on Earth as well as in microgravity showed X-ray diffraction up to 1.15 A. The intensity/sigma ratio was slightly higher for microgravity grown crystals. Crystals grown in agarose gel, both in microgravity and on ground, showed a comparable diffraction power, with a resolution limit of 1.45 A.

Collagen↗