Biomedical subjects

Raul Perez-Jimenez

Publications and source records attributed to Raul Perez-Jimenez.

8 recordsLinked to original sources

PubMed2006

Mechanical unfolding pathways of the enhanced yellow fluorescent protein revealed by single molecule force spectroscopy.

We used single molecule force spectroscopy to characterize the mechanical stability of the enhanced yellow fluorescent protein (EYFP) (a mutant form of the green fluorescent protein (GFP)) and two of its circularly permutated variants. In all three constructs, we found two main unfolding peaks; the first corresponds to a transition state placed close to the termini and the second to a transition state placed halfway through the molecule. We attribute the second transition state to the shear rupture of the beta1- and beta6-strands, which we verified by introducing a point mutation in this region. Although both unfolding peaks were observed in all three EYFP variants, their relative frequency of occurrence varied. Our results demonstrated that the mechanical unfolding pathways in EYFP could be deciphered through the use of circular permutation.

Animals↗

PubMed2006

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments.

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.

Amino Acid Sequence↗

PubMed2005

A simple tool to explore the distance distribution of correlated mutations in proteins.

The analysis of correlated mutations in protein sequence alignments is of considerable interest, since it may provide useful energetic and even structural information (ideally, residue contacts). However, a number of recent experimental studies support the existence of long-distance communication in proteins, a fact that may lead to correlation between distant residues. We introduce in this work a simple statistical procedure to describe the relation structure--alignments on the basis of the residue--residue distance dependence of the number of residue couples over given thresholds of a correlation measure (such as a covariance value). This procedure may lead to clear pictures of the distance distribution of correlated mutations and may provide a simple but efficient tool to explore the different structural features that are reflected in the sequence alignments.

Amino Acids↗

PubMed2005

A stability pattern of protein hydrophobic mutations that reflects evolutionary structural optimization.

We have determined the effect of mutations involving isoleucine and valine (i.e., mutations I-->V and V-->I) on the stability of Escherichia coli thioredoxin. Despite the similarity in chemical structure (V and I differ only in a methyl group), we find that many environments are optimized to a significant extent for either V or I. We find, furthermore, that a plot of effect of hydrophobic mutations on stability versus packing density shows a strikingly simple pattern that clearly reflects evolutionary structural optimization. The existence of such patterns suggests the possibility of rationalizing (and perhaps even predicting) mutation effects on protein stability on the basis of evolutionary models. By "evolutionary model" we specifically refer in this context to a model for mutation effects on stability in which certain physical features of the mutated residue environments are evaluated from an assumption regarding how such environments have been selected during protein evolution (as opposed to a purely "physical model" in which those features would be derived from some kind of energetics analysis of the protein structural characteristics). To illustrate this novel approach and provide general guidelines for its application, we develop here a simple evolutionary model that successfully explains the effect of the I<-->V mutations on thioredoxin stability.

Algorithms↗

PubMed2004

The effect of charge-introduction mutations on E. coli thioredoxin stability.

Technological applications of proteins are often hampered by their low-stability and, consequently, the development of procedures for protein stabilization is of considerable biotechnological interest. Here, we use simple electrostatics to determine positions in E. coli thioredoxin at which mutations that introduce new charged residues are expected to lead to stability enhancement. We also obtain the corresponding mutants and characterize their stability using differential scanning calorimetry. The results are interpreted in terms of the accessibility in the native structure of the mutated residues and the potential effect of the mutations on the residual structure of the denatured state.

Calorimetry, Differential Scanning↗

PubMed2004

Empirical parametrization of pK values for carboxylic acids in proteins using a genetic algorithm.

Considerable effort has been devoted to the development of theoretical electrostatic methods to predict the pK values of ionizable residues in proteins. However, predictions appear often to be still at the qualitative or semi-quantitative level. We believe that, with the increasing number experimentally available pK values for proteins of known structure, an alternative approach becomes feasible: the empirical parametrization of the experimental protein pK database. Of course, in the long term, this empirical approach is no substitute for rigorous electrostatic analysis but, in the short term, it may prove to have useful predictive power and it may help to pinpoint the main structural determinants of pK values in proteins. Here we demonstrate the feasibility of the parametrization approach by fitting (using a genetic algorithm as fitting tool) the database for carboxylic acid pK values in proteins on the basis of an empirical equation that takes into account the two following kinds of effects: (1) long-range charge-charge interactions; (2) interactions of the given carboxylic acid group with its environment in the protein, which are described in terms of contributions from the different kind of atoms present in the protein (atomic contributions).

Algorithms↗

PubMed2004

Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations.

Native proteins are marginally stable. Low thermodynamic stability may actually be advantageous, although the accumulation of neutral, destabilizing mutations may have also contributed to it. In any case, once marginal stability has been reached, it appears plausible that mutations at non-constrained positions become fixed in the course of evolution (due to random drift) with frequencies that roughly reflect the mutation effects on stability ("pseudo-equilibrium hypothesis"). We have found that all glutamate-->aspartate mutations in wild-type Escherichia coli thioredoxin are destabilizing, as well as most of the aspartate-->glutamate mutations. Furthermore, the effect of these mutations on thioredoxin thermodynamic stability shows a robust correlation with the frequencies of occurrence of the involved residues in several-hundred sequence alignments derived from a BLAST search. These results provide direct and quantitative experimental evidence for the pseudo-equilibrium hypothesis and should have general consequences for the interpretation of mutation effects on protein stability, as they suggest that residue environments in proteins may be optimized for stabilizing interactions to a remarkable degree of specificity. We also provide evidence that such stabilizing interactions may be detected in sequence alignments, and briefly discuss the implications of this possibility for the derivation of structural information (on native and denatured states) from comparative sequence analyses.

Aspartic Acid↗

PubMed2004

The efficiency of different salts to screen charge interactions in proteins: a Hofmeister effect?

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces.

Algorithms↗