Enric Querol

Essential catalytic role of Glu134 in endo-β-1,3-1,4-d-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis

Site‐directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo‐β‐1,3‐1,4‐d‐glucan 4‐glucanohydrolase (β‐glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinan: E. coli and their kinetics analysed with barley β‐glucan. Replacement of Glu134 by Gin produced a mutant (E134Q) that retains less than 0.3% of the wild‐type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild‐type indicative of their participation in substrate binding and transition‐state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme‐like mechanism for this family of Bacillus β‐glucan hydrolases with Glu134 being the essential acid catalyst.

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a λ max of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color‐controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.

Fourier Transform Infrared Evidence for Early Deprotonation of Asp85 at Alkaline pH in the Photocycle of Bacteriorhodopsin Mutants Containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered λ max of 555 nm and pK a of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.

Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro91 and Pro186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.

Cloning, expression and purification of human epidermal growth factor using different expression systems.

Epidermal growth factor (EGF) is a protein that belongs to the family of growth factors that bind the ErbB receptors, which play a prominent role in the development of carcinomas. We had demonstrated that potato carboxypeptidase inhibitor (PCI) acts as an EGF antagonist. Because of the low affinity of PCI for the epidermal growth factor receptor, it was decided to design EGF mutants with PCI abilities. In order to achieve this we have first cloned, expressed and purified the native protein, EGF. Different expression systems with different locations of the recombinant protein were designed and a purification protocol was designed with those which allowed expression of EGF. Finally, the sample needed folding. Differences in the amount of EGF obtained and its activity were observed depending on the expression system used.

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin.

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane α-helices, as compared to α-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pKa and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cγ and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.

X-ray absorption and molecular dynamics study of cation binding sites in the purple membrane

The present work describes the results of a study aimed at identifying candidate cation binding sites on the extracellular region of bacteriorhodopsin, including a site near the retinal pocket. The approach used is a combined effort involving computational chemistry methods (computation of cation affinity maps and molecular dynamics) together with the Extended X‐Ray Absorption Fine Structure (EXAFS) technique to obtain relevant information about the local structure of the protein in the neighborhood of Mn2+ ions in different affinity binding sites. The results permit the identification of a high‐affinity binding site where the ion is coordinated simultaneously to Asp212− and Asp85−. Comparison of EXAFS data of the wild type protein with the quadruple mutant E9Q/E74Q/E194Q/E204Q at pH 7.0 and 10.0 demonstrate that extracellular glutamic acid residues are involved in cation binding. Proteins 2007

Coupling between the retinal thermal isomerization and the Glu194 residue of bacteriorhodopsin.

Glu194 is a residue located at the end of F helix on the extracellular side of the light‐induced proton pump bacteriorhodopsin (BR). Currently, it is well recognized that Glu194 and Glu204 residues, along with water clusters, constitute the proton release group of BR. Here we report that the replacement of Glu194 for Gln affects not only the photocycle of the protein but also has tremendous effect on the all‐trans to 13‐cis thermal isomerization. We studied the pH dependence of the dark adaptation of the E194Q mutant and performed HPLC analysis of the isomer compositions of the light‐ and partially dark‐adapted states of the mutant at several pH values. Our data confirmed that E194Q exhibits extremely slow dark adaptation over a wide range of pH. HPLC data showed that a significantly larger concentration of all‐trans isomer was present in the samples of the E194Q mutant even after prolonged dark adaptation. After 14 days in the dark the 13‐cis to all‐trans ratio was 1:3 in the mutant, compared to 2:1 in the wild type. These data clearly indicate the involvement of Glu194 in control of the rate of all‐trans to 13‐cis thermal isomerization.

Molecular dynamics as a tool to help design mutants

A 200 ps MD trajectory of wild type PCI and a 120 ps one for the Pro36Gly putative mutant are studied and compared with the structure of PCI in its complex with carboxypeptidase A (CPA). It is first established that the structures of PCI from X-ray and from MD simulation are essentially equal. Thereafter, data from the PCI-CPA and average MD structures together with available biochemical information are used to identify possible structural factors that may determine the inhibitory power of PCI. These structural determinants are used to analyze the mutant structure. The fold of the mutant protein shows a large degree of stability. The N-terminal tail in PCIm differs from the X-ray structure as it does in PCIw, while the mutants C-terminal tail (which is the primary binding site with CPA) and residues 13–17 present deviations. Differences in fluctuation patterns exist between PCIm and PCIw in residues 2–4 (the N-terminal tail), 13–17, 22–23, 28–81 (the secondary contact site with CPA), and 37–38 (the C-terminal tail); the latter region is rigidified in PCIm. Results show that the MD method is able to sense long-range as well as local perturbative effects produced by amino-acid substitutions in flexible regions of this protein. The simulations suggest that the conformation of the C-terminal tail is less favorable for interaction with the target protein in the mutant than it is in the wild type protein. The Pro-36-Gly mutant is predicted to be a less potent inhibitor.