Maria Teresa Neves Petersen

What distinguishes an esterase from a lipase: a novel structural approach.

Esterases and lipases both hydrolyse ester bonds. Whereas the lipases display high activity towards the aggregated state of its substrate, the esterases typically show highest activity towards the soluble state of its substrate. We have compared the amino acid sequence, the 3D-structure as well as the pH-dependent electrostatic signature of selected members of the two families, for which 3D-structural information is publicly available. Lipases display a statistically significant enhanced occurrence of non-polar residues close to the surface, clustering around the active-site. Lid opening appears to strengthen this pattern further. As we have proposed earlier the active site of lipases displays negative potential in the pH-range associated with their maximum activity, typically at pH values above 8. The esterases show a very similar pattern, however, at pH values around 6 correlated with their usually lower pH-activity optimum.

Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol.

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280-300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri-N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.

Protein Engineering the Surface of Enzymes

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Surface and electrostatics of Cutinases

Publisher Summary The surface of a protein constitutes the interface through which the protein senses the environment surrounding it. Therefore, it should be central to any study aiming at a better understanding of the molecular basis for the interaction between an enzyme and its substrate or inhibitor, a receptor and its ligand, or any other type of molecular recognition. In the study of biological macromolecular systems it is becoming increasingly evident that electrostatic interactions contribute significantly to folding, conformational stability, enzyme activity, and binding energies as well as to protein-protein interactions. This chapter presents approaches to modeling electrostatic interactions in biomolecular systems. It describes an approach for the calculation of the pk a values of titratable groups in proteins. The chapter also presents some methods that can be used to map and study the amino acid distribution on the molecular surface of proteins. The combination of graphic visualization of the electrostatic fields with the knowledge about the location of key residues on the protein surface allows envisioning atomic models for enzyme function. Some of these methods are applied to the enzymes of the cutinase family.

Identification of important motifs in protein sequences: program MULTIM and its applications to lipase-related sequences.

Publisher Summary This chapter presents a novel methodology called MULTIM (multiple motifs); it is based on a combination of graphic visualization of the results and a simple, yet versatile, method for the identification of conserved motifs in the protein sequences being compared. Its true value resides in its ability to present the results in an easy-to-grasp graphic picture that, in addition, is well suited for documentation and/or presentation purposes. The chapter illustrates the qualities of MULTIM on the lipase and esterase family of sequences, as well as other selected sequences. The chapter discusses the present limitations of MULTIM. In its present version, MULTIM presents graphically the alignment of motifs found by MULTIM. Because motifs contain at the least two residues each, and most often three or more, the graphic alignment is intrinsically coarse grained. If it is essential that the alignment be completed at the single-residue level, the user will have to do it manually.