My research interests are devoted to investigate the physicochemical basis underlying protein’s structure-function relationship and protein-protein interactions and how this knowledge can be applied to obtain fundamental insight in complex systems of biomedical relevance.
Hence, combining expertise in biophysics, biochemistry, molecular and cellular biology, a research program with a multidisciplinary approach is developed focused on two main areas:
1. Protein structure, stability and folding.
On this subject a comparative study of the temperature and pressure effect on the structure, stability and folding/unfolding pathways has been performed using several physical-chemical techniques (UV, CD, NMR, X-Ray) combined with protein engineering.
Research in this field has contributed to deepen our knowledge in the following specific aims:
a. To characterize the transition state of protein unfolding induced by pressure.
b. To better understand protein’s unfolding-folding pathways using heating and cooling temperature-jumps.
c. To provide fundamental insight into the oligomerization process and the contribution of specific substitutions on protein stability, by solving the 3D-structures of single protein variants and oligomers at atmospheric and high pressure.
2. Molecular basis of the biological activities of cytotoxic proteins.
Among the biological activities presented by some ribonucleases, the selective cytotoxicity for tumour cells is of exceptional interest. A deeper understanding of the molecular basis that endow a ribonuclease with cytotoxic properties can help to improve the abilities these toxins, and in the design of new drugs that can be used as therapeutic agents.
In this area we have contributed to better understand and to develop the following issues:
a. To characterize the internalization pathway of onconase, a selective cytotoxic protein.
b. To endow human pancreatic ribonuclease (HP-RNase) with cytotoxic activity.
c. To design engineered ribonucleases with ‘de novo’ acquired antimicrobial activity and zymogens based on onconase and HP-RNase scaffolds that can be activated by HIV-1 protease.
More recently, my attention has focused to technologies such as expressed protein ligation (EPL) and protein trans splicing (PTS) which allow synthetic peptides and recombinant proteins to be chemo-selectively linked together.
The former approach opens up protein’s research to the tools of organic chemistry by allowing the selective and homogeneous incorporation of unnatural aminoacids, posttranslational modifications or isotopic probes into specific protein sites. Modifications incorporated into proteins by this strategy may provide more accurate information about how proteins work than the classical protein engineering approach.
Protein splicing is a posttranslational event by which an intervening sequence – termed an intein - catalyzes its removal from a host protein, the extein. In PTS the intein is split into two pieces and splicing occurs only upon reconstitution of both fragments. PTS is an alternative to EPL for the semisynthesis of proteins in vitro and in vivo. In addition, PTS can be used to develop strategies to control protein’s function in vivo. To trigger posttranslational synthesis of a target protein in a tunable manner would provide us with the control of protein function both temporally and spatially. This approach is expected to have widespread applications for studying protein function in the postgenomic era.