For more details and published work you may visit  the web of the Protein Folding and Stability and Molecular Design Group.

Stability and stabilization of non two-state proteins

The stability of a protein is the free energy difference between the native, functional conformation and the unfolded state.  Much we have learned on protein stability principles and on protein stabilization by studying small 2-state proteins (whose molecules can only be either fully folded or fully unfolded).  However, most proteins (perhaps as many as 90 % of the human proteome) are large and complex and may adopt under certain conditions intermediate conformations that are neither native nor fully unfolded.  For these proteins it is useful to distinguish between the "relevant" and the "residual" components of the overall stability.  We are investigating whether protein engineering approaches .

developed in many labs. to increase the overall stability of small two-state proteins are useful to increase the relevant stability of complex proteins. Colaborating groups: JM Sánchez-Ruiz (Granada), MA Jiménez (Madrid).

Prediction of mutational effects on protein stability

The stability of proteins can be substantially altered (decreased or increased) by point mutations.  We try to understand the Physics determining the effects of mutations on protein stability so that we can eventually achieve a quantitative understanding leading to predictive power. We are specially interested in internal hydrophobic/van der Waals interactions, hydrogen bonds and a-helix energetics.  Our approach combines experimental analysis of protein and peptide stability with theoretical calculations and parameterizations based on our own data and on data available in the scientific literature. Collaborating groups: Carlos J. Camacho (Boston).

Screening and design of protein ligands and inhibitors

The activity and stability of proteins can be modulated by ligand binding.  Designing or identifying protein ligands is a need of both Medicine and Industry and may be of help in order to understand the complex molecular interaction networks operating in living beings.  We develop methodology for massive virtual screening of large libraries of potential drugs and for massive screening of real libraries of chemical compounds.  One current objective is identifying inhibitors of the flavodoxin of Helicobacter pilory, which has been shown to be essential for the survival of the pathogen.

Folding diseases

Many human diseases have been recently recognised as arising from point mutations that give rise to unstable or aggregation prone proteins that are either unable to perform their physiological function or whose aggregates are pernicious.  We are investigating a very common folding disease (human hypercolesterolemia) that arises from mutations in the LDL receptor that destabilise the modules responsible for interacting with the circulating lipoproteins.  Our approach includes expression and characterisation of LDL binding modules carrying mutations related to the disease, Molecular Dynamics simulations of mutated modules and screening for stabilising ligands. Collaborating groups: F. Falo (Zaragoza), M- Pocoví (Zaragoza), JC Rodríguez (Santander).

Kinetic and thermodynamic determinants of protein/cofactor recognition

The affinity of protein/cofactor complexes is a reflection of the interactions established between protein, cofactor and water atoms and can be investigated by a combination of thermodynamic measurements and Protein Engineering.  An additional issue is that of how the protein and cofactor molecules recognise each other and form the functional complex as the molecules approach in solution.  Both aspects of protein/cofactor interactions are being actively investigated using flavodoxin, from which the FMN cofactor can easily removed and added back.  Collaborating groups: Carlos Gómez-Moreno and M. Medina (Zaragoza)

Structural determination of transition states of protein folding and of equilibrium intermediates

X-ray and NMR are usually the techniques that provide structural information on proteins.  There are, nevertheless, some elusive protein conformations (intermediates, transition states of folding) for which X-ray or NMR may not be appropriate.  Protein intermediates and transition states are

nevertheless very  important to understand how proteins fold and how they become partly or fully denatured.  We have recently extended Phi-analysis to determine low resolution structures of equilibrium protein folding intermediates.  Our Phi-structure of the apoflavodoxin thermal equilibrium intermediate shows a good correspondence with available nmr data and is being used in redesigning the stability of the native state relative to the intermediate.  We are currently investigating the structure of the transition state of folding  pf apoflavodoxin.

These are some representative publications.

• The "relevant" stability of proteins with equilibrium intermediates (2002) The Scientific World 2:1209-1215 PDF

• Predicting the structure of protein cavities created by mutation (2002) Protein Engineering 11:669-675 PDF

• Four-state equilibrium unfolding of an scFv antibody fragment (2002) Biochemistry 41:9873-9884 PDF

• An Intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA (2003) Human Molecular Genetics 12:329-339 PDF               

• How FMN binds to Anabaena apoflavodoxin: a hydrophobic encounter at an open binding site (2003) J. Biol. Chem. 278: 24053-24061 PDF

• The long and short flavodoxins. I: role of the differentiating loop in apoflavodoxin structure and FMN binding (2004) J. Biol. Chem. 279: 47177-47183 PDF            

• Do proteins always benefit from a stability increase? Relevant and residual stabilization in a three-state protein by charge optimization (2004) J. Mol.Biol. 344:223-237 PDF 

• Structure of stable protein folding intermediates by equilibrium F‑analysis: the apoflavodoxin thermal intermediate (2004) J. Mol.Biol. 344:239-255 PDF  

• Towards a new therapeutic target: Helicobacter pylori flavodoxin (2005) Biophys. Chem.  In Press

• A double-deletion method to quantifying incremental binding energies in proteins from experiment.  Example of a destabilizing hydrogen bonding pair. (2005) Biophys. J. In Press

These are some tutorials or general references on protein stability and folding.

In English VIDEO:How do proteins fold: Folding at Home:EDUCATION Protein Structure Prediction Centre Molecular Simulations of Proteins And Peptides Overview of molecular forces in relation to protein structure

In Spanish La estabilidad de las proteínas  El plegamiento de las proteínas Estabilidad y plegamiento de proteínas y la interacción proteína/ligando Estructura de proteínas. Ed. Ariel

Click to send mails