Biomedical subjects

Javier Sancho

Publications and source records attributed to Javier Sancho.

25 records · Page 2Linked to original sources

PubMed2003

Different thermostability of skeletal muscle glyceraldehyde-3-phosphate dehydrogenase from hibernating and euthermic jerboa (Jaculus orientalis).

In previous study, we demonstrated that the specific activity of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in skeletal muscle of induced hibernating jerboa (hibernating GAPDH) was 3 4 folds lower than that of the one in the skeletal muscle of the euthermic jerboa (euthermic GAPDH). A significant decrease in both GAPDH protein and GapC mRNA levels occurs when hibernating, but the purified hibernating GAPDH is less active than the euthermic GAPDH. To investigate the physico-chemical basis of this lower activity, the behaviour during thermal inactivation of skeletal muscle GAPDH from hibernating and euthermic tissues was examined by a variety of spectroscopic techniques, including fluorescence emission, circular dichroism and ultraviolet absorption. A clear resistance to thermal denaturation was observed in the hibernating GAPDH compared with the euthermic GAPDH. The different temperature of denaturation found in these proteins by both fluorimetry and circular dichroism indicates that there might exist conformational changes of GAPDH upon hibernation that could affect the stability of this enzyme.

Animals↗

PubMed2002

Four-state equilibrium unfolding of an scFv antibody fragment.

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.

Circular Dichroism↗

PubMed2002

The 'relevant' stability of proteins with equilibrium intermediates.

Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the 'relevant' stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the 'relevant' and 'nonrelevant' terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented.

Computational Biology↗

PubMed2002

Anabaena sp. PCC 7119 flavodoxin as electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of Trp(57) and Tyr(94).

The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp. PCC 7119 in complex formation and electron transfer (ET) with its natural partners, photosystem I (PSI) and ferredoxin-NADP(+) reductase (FNR), was examined in mutants of the key residues Trp(57) and Tyr(94). The mutants' ability to form complexes with either FNR or PSI is similar to that of wild-type Fld. However, some of the mutants exhibit altered kinetic properties in their ET processes that can be explained in terms of altered flavin accessibility and/or thermodynamic parameters. The most noticeable alteration is produced upon replacement of Tyr(94) by alanine. In this mutant, the processes that involve the transfer of one electron from either PSI or FNR are clearly accelerated, which might be attributable to a larger accessibility of the flavin to the reductant. However, when the opposite ET flow is analyzed with FNR, the reduced Y94A mutant transfers electrons to FNR slightly more slowly than wild type. This can be explained thermodynamically from a decrease in driving force due to the significant shift of 137 mV in the reduction potential value for the semiquinone/hydroquinone couple (E(1)) of Y94A, relative to wild type (Lostao, A., Gómez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344). The behavior of the rest of the mutants can be explained in the same way. Overall, our data indicate that Trp(57) and Tyr(94) do not play any active role in flavodoxin redox reactions providing a path for the electrons but are rather involved in setting an appropriate structural and electronic environment that modulates in vivo ET from PSI to FNR while providing a tight FMN binding.

Anabaena↗

PubMed2002

Predicting the structure of protein cavities created by mutation.

To assist in the efficient design of protein cavities, we have developed a minimization strategy that can predict with accuracy the fate of cavities created by mutation. We first modelled, under different conditions, the structures of six T4 lysozyme and cytochrome c peroxidase mutants of known crystal structure (where long, hydrophobic, buried side chains have been replaced by shorter ones) by minimizing the virtual structures derived from the corresponding wild-type co-ordinates. An unconstrained pathway together with an all-atom atom representation and a steepest descent minimization yielded modelled structures with lower root mean square deviations (r.m.s.d) from the crystal structures than other conditions. To test whether the method developed was generally applicable to other mutations of the kind, we have then modelled eighteen additional T4 lysozyme, barnase and cytochrome c peroxidase mutants of known crystal structure. The models of both cavity expanding and cavity collapsing mutants closely fit their crystal structures (average r.m.s.d. 0.33 +/- 0.25 A, with only one poorer prediction: L121A). The structure of protein cavities generated by mutation can thus be confidently simulated by energy minimization regardless of the tendency of the cavity to collapse or to expand. We think this is favoured by the fact that the typical response observed in these proteins to cavity-creating mutations is to experience only a limited rearrangement.

Amino Acid Substitution↗

PubMed2002

Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects.

Electrostatic contributions to the conformational stability of apoflavodoxin were studied by measurement of the proton and salt-linked stability of this highly acidic protein with urea and temperature denaturation. Structure-based calculations of electrostatic Gibbs free energy were performed in parallel over a range of pH values and salt concentrations with an empirical continuum method. The stability of apoflavodoxin was higher near the isoelectric point (pH 4) than at neutral pH. This behavior was captured quantitatively by the structure-based calculations. In addition, the calculations showed that increasing salt concentration in the range of 0 to 500 mM stabilized the protein, which was confirmed experimentally. The effects of salts on stability were strongly dependent on cationic species: K(+), Na(+), Ca(2+), and Mg(2+) exerted similar effects, much different from the effect measured in the presence of the bulky choline cation. Thus cations bind weakly to the negatively charged surface of apoflavodoxin. The similar magnitude of the effects exerted by different cations indicates that their hydration shells are not disrupted significantly by interactions with the protein. Site-directed mutagenesis of selected residues and the analysis of truncation variants indicate that cation binding is not site-specific and that the cation-binding regions are located in the central region of the protein sequence. Three-state analysis of the thermal denaturation indicates that the equilibrium intermediate populated during thermal unfolding is competent to bind cations. The unusual increase in the stability of apoflavodoxin at neutral pH affected by salts is likely to be a common property among highly acidic proteins.

Apoproteins↗

PubMedDate not supplied

Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations.

Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that Miglustat is not only a substrate reducer but also an inhibitor of the GC degradation, with very promising clinical implications for the treatment of GD patients.

1-Deoxynojirimycin↗