Evamaria I. Petersen

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light‐exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV‐induced changes in three‐dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon‐induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two‐state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH‐dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV‐induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far‐reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.

EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, d‐alanyl‐d‐alanine‐peptidases (DD‐peptidases) and β‐lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of β‐lactamases and DD‐peptidases. The chemical reactivities of esters and β‐lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying β‐lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD‐peptidase and class C β‐lactamases. Site‐directed mutagenesis and the crystal structure of the complex with diisopropyl‐fluorophosphate suggest Ser75 within the “β‐lactamase” Ser‐x‐x‐Lys motif to act as catalytic nucleophile. Despite its structural homology to β‐lactamases, EstB shows no β‐lactamase activity. Although the nature and arrangement of active‐site residues is very similar between EstB and homologous β‐lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzymes selectivity for ester hydrolysis versus β‐lactam cleavage.

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.

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase.

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 105 M−1). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X‐ray crystallography in its apo form and after cocrystallization with diethyl p‐nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid.

In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.

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.