Javier Santos

Correction for phylogeny, small number of observations and data redundancy improves the identification of coevolving amino acid pairs using mutual information

MOTIVATIONnMutual information (MI) theory is often applied to predict positional correlations in a multiple sequence alignment (MSA) to make possible the analysis of those positions structurally or functionally important in a given fold or protein family. Accurate identification of coevolving positions in protein sequences is difficult due to the high background signal imposed by phylogeny and noise. Several methods have been proposed using MI to identify coevolving amino acids in protein families.nnnRESULTSnAfter evaluating two current methods, we demonstrate how the use of sequence-weighting techniques to reduce sequence redundancy and low-count corrections to account for small number of observations in limited size sequence families, can significantly improve the predictability of MI. The evaluation is made on large sets of both in silico-generated alignments as well as on biological sequence data. The methods included in the analysis are the APC (average product correction) and RCW (row-column weighting) methods. The best performing method was APC including sequence-weighting and low-count corrections. The use of sequence-permutations to calculate a MI rescaling is shown to significantly improve the prediction accuracy and allows for direct comparison of information values across protein families. Finally, we demonstrate how a lower bound of 400 sequences <62% identical is needed in an MSA in order to achieve meaningful predictive performances. With our contribution, we achieve a noteworthy improvement on the current procedures to determine coevolution and residue contacts, and we believe that this will have potential impacts on the understanding of protein structure, function and folding.

Structural selection of a native fold by peptide recognition. Insights into the thioredoxin folding mechanism.

Thioredoxins (TRXs) are monomeric alpha/beta proteins with a fold characterized by a central twisted beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix 5 of TRX against the remainder of the protein involves a close packing of hydrophobic surfaces, offering the opportunity of studying a fine-tuned molecular recognition phenomenon with long-range consequences on the acquisition of tertiary structure. In this work, we focus on the significance of interactions involving residues L94, L99, E101, F102, L103 and L107 on the formation of the noncovalent complex between reduced TRX1-93 and TRX94-108. The conformational status of the system was assessed experimentally by circular dichroism, intrinsic fluorescence emission and enzymic activity; and theoretically by molecular dynamics simulations (MDS). Alterations in tertiary structure of the complexes, resulting as a consequence of site specific mutation, were also examined. To distinguish the effect of alanine scanning mutagenesis on secondary structure stability, the intrinsic helix-forming ability of the mutant peptides was monitored experimentally by far-UV CD spectroscopy upon the addition of 2,2,2-trifluoroethanol, and also theoretically by Monte Carlo conformational search and MDS. This evidence suggests a key role of residues L99, F102 and L103 on the stabilization of the secondary structure of alpha-helix 5, and on the acquisition of tertiary structure upon complex formation. We hypothesize that the transition between a partially folded and a native-like conformation of reduced TRX1-93 would fundamentally depend on the consolidation of a cooperative tertiary unit based on the interaction between alpha-helix 3 and alpha-helix 5.

Molecular Basis of Hydroperoxide Specificity in Peroxiredoxins: The Case of AhpE from Mycobacterium tuberculosis

Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzymes dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.

Replacement of methionine‐161 with threonine eliminates a major by‐product of human glutamate decarboxylase 65‐kDa variant expression in Escherichia coli

Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell: Metabolic control analysis and the glycolytic pathway

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde‐3‐phosphate‐dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low‐cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi‐well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course.