Tubert P.,Laboratori Denginyeria Of Proteines |
Tubert P.,Institute Dinvestigacio Biomdica Of Girona Idibgi |
Laurents D.V.,CSIC - Institute of Physical Chemistry "Rocasolano" |
Ribo M.,Laboratori Denginyeria Of Proteines |
And 6 more authors.
Biophysical Journal | Year: 2011
The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr 25, Gln 101, and Pro 19, with Asn 17, Ser 21, and Ser 23, are found to be the most significant; notably, Glu 103 and Arg 104, which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization. © 2011 Biophysical Society.
Ercole C.,University of Naples Federico II |
Lopez-Alonso J.P.,CSIC - Institute of Physical Chemistry "Rocasolano" |
Font J.,Laboratori Denginyeria Of Proteines |
Font J.,CENTENARY INSTITUTE |
And 4 more authors.
Archives of Biochemistry and Biophysics | Year: 2011
RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A "C-dimer", the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes. © 2010 Elsevier Ltd. All rights reserved.
Vert A.,Laboratori Denginyeria Of Proteines |
Vert A.,Institute Dinvestigacio Biomedica Of Girona Dr Josep Trueta Idibgi |
Castro J.,Laboratori Denginyeria Of Proteines |
Castro J.,Institute Dinvestigacio Biomedica Of Girona Dr Josep Trueta Idibgi |
And 12 more authors.
Molecular Pharmaceutics | Year: 2012
Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5. © 2012 American Chemical Society.