Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 1, 2000, pp. 53

Javier Sancho Sanz

Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza. 50009-Zaragoza. Spain. E-mail: jsancho@posta.unizar.es URL: http://wwwbi oq.unizar.eslwwwbioqespanol/JSS.htm

Introduction

Proteins are linear polymers that fold into compact conformations in order to acquire the particular shape that confers them useful biological properties. The native conformations of proteins are stabilized by intra protein interactions (hydrogen bonds, van der Waals, charge/charge, charge/dipole, charge/p and p/p) and by the hydrophobic effect, and they are destabilized mainly by the huge conformational entropy change of folding. A delicate balance between all those factors determines that the average stability of proteins is low (5-15 kcal mol-1). Although 5 kcal mol is enough to ensure that more than 99% of the protein molecules are in the native conformation at any time, the equilibrium between native and denatured conformations forces all the molecules to be unfolded now and then. In the absence of side reactions this is not a problem, but the unfolded state and, perhaps even more, some conformations intermediate between the native and the unfolded state can experience aggregation and other reactions leading to an irreversibly inactivated protein. Irreversible inactivation from the folded conformation is also possible but usually less severe. Since the unfolded state has a greater tendency to irreversible inactivation, one way to minimize these reactions is to reduce the concentration of unfolded molecules. This can be easily done by increasing the conformational stability of proteins.

Materials and Methods

The conformational stability of a protein can be measured by chemical denaturation (usually with urea or guanidinium hydrochoride) or by thermal denaturation [1]. Amino acid residues of a protein can be replaced at will if the gene is cloned. The rational replacement of residues requires knowledge of the tridimensional structure (by X-ray or NMR).

Results and Discussion

There are plenty of possible strategies to rationally stabilize proteins and many have been tested and shown to work fine in many cases. Introduction ion of new disulfide bridges, engineering of charge/dipole interactions, decreasing the entropy of the denatured state by removing glicines or introducing prolines [2], removing hyperexposed hydrophobic residues, engineering metal binding sites, etc. As an example, we have recently shown that neutral hydrogen bonds may sometimes be more stable than charged ones [3]. Based on this, we have stabilized our model protein apoflavodoxin by neutralizing solvent exposed hydrogen bonds by site directed mutagenesis.

Stabilizing proteins may be important for biotechnological purposes as it may increase the operational life of the valuable protein. This is, however, not the only interesting application, as there is now growing evidence of many diseases being caused by protein misfolding, such as amyloid diseases and certain cancers [4]. The latter case could be a simple matter of insufficient stability of a protein that regulates the cell cycle leading to malfunction and cell proliferation.

Rational protein stabilization is thus an important, yet not very used strategy, that may have a great impact both in industry and in medicine.

References

1. Pace CN, Shirley BA, Thomson JA. Measuring the conformational stability of a protein. In Creighton TE Ed. Protein structure A practical approach. Oxford- IRL Press. 1989;pp 311–30.

2. Branden C, Tooze J. Introduction to Protein Structure 2nd ed. Garland Pub., New York 1998.

3. Fernández-Recio J, Romero A, Sancho J. Energetics of a hydrogen bond (charged and neutral) and of a cation-p interaction in apoflavodoxin. J. Mol. Biol 1999;290:319–31.

4. Radford SE, Dobson CM. From computer simulations to human disease- emerging themes in protein folding. Cell 1999;97:291–8.

Copyright 2000 Elfos Scientiae