Stable monomers in the ancestral sequence reconstruction of the last opisthokont common ancestor of dimeric triosephosphate isomerase.

Function and structure are strongly coupled in obligated oligomers such as Triosephosphate isomerase (TIM). In animals and fungi, TIM monomers are inactive and unstable. Previously, we used ancestral sequence reconstruction to study TIM evolution and found that before these lineages diverged, the last opisthokonta common ancestor of TIM (LOCATIM) was an obligated oligomer that resembles those of extant TIMs. Notably, calorimetric evidence indicated that ancestral TIM monomers are more structured than extant ones. To further increase confidence about the function, structure, and stability of the LOCATIM, in this work, we applied two different inference methodologies and the worst plausible case scenario for both of them, to infer four sequences of this ancestor and test the robustness of their physicochemical properties. The extensive biophysical characterization of the four reconstructed sequences of LOCATIM showed very similar hydrodynamic and spectroscopic properties, as well as ligand-binding energetics and catalytic parameters. Their 3D structures were also conserved. Although differences were observed in melting temperature, all LOCATIMs showed reversible urea-induced unfolding transitions, and for those that reached equilibrium, high conformational stability was estimated (ΔGTot = 40.6-46.2 kcal/mol). The stability of the inactive monomeric intermediates was also high (ΔGunf = 12.6-18.4 kcal/mol), resembling some protozoan TIMs rather than the unstable monomer observed in extant opisthokonts. A comparative analysis of the 3D structure of ancestral and extant TIMs shows a correlation between the higher stability of the ancestral monomers with the presence of several hydrogen bonds located in the "bottom" part of the barrel.

High Interfacial Viscoelasticity of Aqueous Mixed Dodecyltrimethylammonium Bromide-Sodium Dodecyl Sulfate Surfactants Forming Inclusion Complexes with α-Cyclodextrin.

Mixtures of anionic-cationic surfactants have shown high synergistic effects in the bulk solution and at the liquid/air interface. These studies have been limited to a reduced concentration range, where there is no formation of aggregates or precipitates. The addition of host molecules, such as cyclodextrins, to these systems reduces the effects of precipitation by forming inclusion complexes and also modifies the values of other surfactant properties, like the Krafft temperature and the critical aggregation concentration (CAC). We studied the interfacial synergistic effects promoted by electrostatic interactions, using the Rosen model to calculate an interaction parameter for mixtures of sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) in the presence of α-cyclodextrin (αCD), in aqueous solutions. We measured the CAC of SDS-DTAB-αCD mixtures using a pendant drop tensiometer, with the αCD concentration fixed at 10 mM and at 283.15 K. We performed rheological measurements on the mixtures where the surfactant total concentration is fixed below the measured CAC, varying the αCD concentration and temperature. We found that the dilatational modulus shows a clear correlation with the interaction parameter. It appears that the attractive interactions within the film are those due to the inclusion complexes formed by two αCD and one surfactant molecule, which according to the previous studies, is the dominant species in both the bulk and liquid/air interface. The synergistic effect observed here for SDS-DTAB surfactant mixtures with αCD can be applied to systems and processes (drop emission, drug delivery methods, stabilization of viral capsids and bacterial membranes, and emulsification) where interfacial processes require specific viscoelastic properties.

ATCUN-like Copper Site in ßB2-Crystallin Plays a Protective Role in Cataract-Associated Aggregation.

Cataract is the leading cause of blindness worldwide, and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce the aggregation of human γ-crystallins. Here, we evaluated the impact of divalent metal ions in the aggregation of human ßB2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of ßB2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating the formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of ßB2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in ßB2-crystallin, one of them with spectroscopic features typical for Cu2+ bound to an amino-terminal copper and nickel (ATCUN) binding motif, which is found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of ßB2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Isothermal titration calorimetry indicates a nanomolar Cu2+ binding affinity for the ATCUN-like site. An N-truncated form of ßB2-crystallin is more susceptible to Cu-induced aggregation and is less thermally stable, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy studies reveal the presence of a copper redox active site in ßB2-crystallin that is associated with metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of ßB2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in ßB2-crystallin plays a functional/protective role or constitutes a vestige from its evolution as a lens structural protein remains to be elucidated.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of ß-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

Dynamical shapes of droplets of cyclodextrin-surfactant solutions.

We present a series of experiments with droplets of aqueous cyclodextrin-surfactant solutions, in which the volume is reduced after the equilibrium spherical shape is reached. The final shape of the drop after this perturbation is found to be dependent on the concentration of inclusion complexes in the bulk of the solution. These inclusion complexes are formed by two cyclodextrin molecules and one surfactat molecule. We propose a model to describe these dynamical processes. Dipole-dipole interactions on the surface of the drop trigger a competition between water surface tension and dipole-dipole interaction energies. The results of the model reproduce the spherical and rod-like shapes found in the experiments.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (ß/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

Dipole-dipole interactions control the interfacial rheological response of cyclodextrin/surfactant solutions.

A recent surface rheological study has shown that aqueous solutions of α-cyclodextrin (αCD) with anionic surfactants (S) display a remarkable viscoelasticity at the liquid/air interface, which has not been observed in similar systems. The dilatational modulus is various orders of magnitude larger than those for the binary mixtures αCD + water and S + water. The rheological response has been qualitatively related to the bulk distribution of species, the 2 : 1 inclusion complexes (αCD2 : S) playing a fundamental role. In this work, we have developed a model that considers dipole-dipole interactions between 2 : 1 inclusion complexes ordered on the liquid/air interface. When the model is applied to the specific experimental conditions, the dependencies on concentration and temperature of the dilatational modulus and the surface tension were found to be in excellent agreement with the data, indicating clearly that dipole-dipole interactions determine and control the rheological behavior of the interface.

Antimicrobial Peptide against Mycobacterium Tuberculosis That Activates Autophagy Is an Effective Treatment for Tuberculosis.

Mycobacterium tuberculosis (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 µM), while increasing the dose (50 µM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.

Comparative assessment of the VH-VL and VL-VH orientations of single-chain variable fragments of scorpion toxin-neutralizing antibodies.

The present study evaluated the effect of the change in the orientation of the VH-VL variable domains to VL-VH on the physicochemical and functional properties of two scorpion toxin-neutralizing scFvs. The results showed that the level of expression of proteins obtained from the periplasm of E. coli is the factor mainly affected, either with an increase or decrease in the amount of protein recovered. Likewise, the functional recognition activity in the presence of a denaturing agent showed slight variations in the two orientations. In contrast, recognition and biological activity (neutralizing capacity) are maintained. At the interaction level, the change marginally modified the kinetic association and dissociation constants without significantly modifying the value of the affinity constants. Similarly, it was observed that the thermodynamic stability of the proteins did not show significant variations either. These results contrast with some reports of the effect of changing the orientation of domains, suggesting that it is not possible to predict which orientation of the variable domains of an scFv is more favorable or if they are equivalent, as in the case of scFvs previously matured by directed evolution techniques.

Highly viscoelastic films at the water/air interface: α-Cyclodextrin with anionic surfactants.

This work showcases the remarkable viscoelasticity of films consisting of α-cyclodextrin (α-CD) and anionic surfactants (S) at the water/air interface, the magnitude of which has not been observed in similar systems. The anionic surfactants employed are sodium salts of a homologous series of n-alkylsulfates (n = 8-14) and of dodecylsulfonate. Our hypothesis was that the very high viscoelasticity can be systematically related to the bulk and interfacial properties of the system. Through resolution of the bulk distribution of species using isothermal titration calorimetry, the high dilatational modulus is related to (α-CD)2:S1 inclusion complexes in the bulk with respect to both the bulk composition and temperature. Direct interfacial characterization of α-CD and sodium dodecylsulfate films at 283.15 K using ellipsometry and neutron reflectometry reveals that the most viscoelastic films consist of a highly ordered monolayer of 2:1 complexes with a minimum amount of any other component. The orientation of the complexes in the films and their driving force for adsorption are discussed in the context of results from molecular dynamics simulations. These findings open up clear potential for the design of new functional materials or molecular sensors based on films with specific mechanical, electrical, thermal, chemical, optical or even magnetic properties.

Thermodynamic Stability of Human γD-Crystallin Mutants Using Alchemical Free-Energy Calculations.

γD-Crystallin (HγDC) is a key structural protein in the human lens, whose aggregation has been associated with the development of cataracts. Single-point mutations and post-translational modifications destabilize HγDC interactions, forming partially folded intermediates, where hydrophobic residues are exposed and thus triggering its aggregation. In this work, we used alchemical free-energy calculations to predict changes in thermodynamic stability (ΔΔG) of 10 alanine-scanning variants and 12 HγDC mutations associated with the development of congenital cataract. Our results show that W42R is the most destabilizing mutation in HγDC. This has been corroborated through experimental determination of ΔΔG employing differential scanning calorimetry. Calculations of hydration free energies from the HγDC WT and the W42R mutant suggested that the mutant has a higher aggregation propensity. Our combined theoretical and experimental results contribute to understand HγDC destabilization and aggregation mechanisms in age-onset cataracts.

AFFINImeter: A software to analyze molecular recognition processes from experimental data.

The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of supramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.

Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function.

Function, structure, and stability are strongly coupled in obligated oligomers, such as triosephosphate isomerase (TIM). However, little is known about how this coupling evolved. To address this question, five ancestral TIMs (ancTIMs) in the opisthokont lineage were inferred. The encoded proteins were purified and characterized, and spectroscopic and hydrodynamic analysis indicated that all are folded dimers. The catalytic efficiency of ancTIMs is very high and all dissociate into inactive and partially unfolded monomers. The placement of catalytic residues in the three-dimensional structure, as well as the enthalpy-driven binding signature of the oldest ancestor (TIM63) resemble extant TIMs. Although TIM63 dimers dissociate more readily than do extant TIMs, calorimetric data show that the free ancestral subunits are folded to a greater extent than their extant counterparts are, suggesting that full catalytic proficiency was established in the dimer before the stability of the isolated monomer eroded. Notably, the low association energy in ancTIMs is compensated for by a high activation barrier, and by a significant shift in the dimer-monomer equilibrium induced by ligand binding. Our results indicate that before the animal and fungi lineages diverged, TIM was an obligated oligomer with substrate binding properties and catalytic efficiency that resemble that of extant TIMs. Therefore, TIM function and association have been strongly coupled at least for the last third of biological evolution on earth. DATABASES: PDB Entry: 6NEE. ENZYMES: Triosephosphate isomerase 5.3.1.1, Glycerol-3-phosphate dehydrogenase 1.1.1.8.

Structure and conformational stability of the triosephosphate isomerase from Zea mays. Comparison with the chemical unfolding pathways of other eukaryotic TIMs.

We studied the structure, function and thermodynamic properties for the unfolding of the Triosephosphate isomerase (TIM) from Zea mays (ZmTIM). ZmTIM shows a catalytic efficiency close to the diffusion limit. Native ZmTIM is a dimer that dissociates upon dilution into inactive and unfolded monomers. Its thermal unfolding is irreversible with a Tm of 61.6 ±â€¯1.4 °C and an activation energy of 383.4 ±â€¯11.5 kJ mol-1. The urea-induced unfolding of ZmTIM is reversible. Transitions followed by catalytic activity and spectroscopic properties are monophasic and superimposable, indicating that ZmTIM unfolds/refolds in a two-state behavior with an unfolding ΔG°(H20) = 99.8 ±â€¯5.3 kJ mol-1. This contrasts with most other studied TIMs, where folding intermediates are common. The three-dimensional structure of ZmTIM was solved at 1.8 Å. A structural comparison with other eukaryotic TIMs shows a similar number of intramolecular and intermolecular interactions. Interestingly the number of interfacial water molecules found in ZmTIM is lower than those observed in most TIMs that show folding intermediates. Although with the available data, there is no clear correlation between structural properties and the number of equilibrium intermediates in the unfolding of TIM, the identification of such structural properties should increase our understanding of folding mechanisms.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability.

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.

Complexation thermodynamics of α-cyclodextrin with ionic surfactants in water.

The interaction of α-cyclodextrin (α-CD) with ten ionic surfactants (S) in water was systematically examined using isothermal titration calorimetry. The S comprised cationic and anionic head groups while the hydrocarbon alkyl chain length varied from eight to fourteen carbon atoms. The heat data were measured at five temperatures ranging from 283.15K to 318.15K and were treated simultaneously allowing the estimation of a thermodynamically consistent temperature dependence of the equilibrium constant, as well as the enthalpy and heat capacity for the sequential formation of the [α-CD·S] and the [α-CD2·S] inclusion complexes. All attempts to fit the data assuming that only [α-CD·S] complexes are present failed. It was found that the thermodynamic footprint of the [α-CD·S] complexes does not depend importantly on the head group, while the formation and stabilization of the [α-CD2·S] complexes is strongly influenced by the chemical nature of the polar head group. Several contributions to the thermodynamic parameters are discussed in detail. Among the studied surfactants, the decyl- and octylsulfates were identified as those with a predominant content of [α-CD2·S] complexes and hence they are promising candidates to form viscoelastic films at the liquid/air interface, as it was found previously for the dodecylsulfate surfactant.

A guide to the effects of a large portion of the residues of triosephosphate isomerase on catalysis, stability, druggability, and human disease.

Triosephosphate isomerase (TIM) is a ubiquitous enzyme, which appeared early in evolution. TIM is responsible for obtaining net ATP from glycolysis and producing an extra pyruvate molecule for each glucose molecule, under aerobic and anaerobic conditions. It is placed in a metabolic crossroad that allows a quick balance of the triose phosphate aldolase produced by glycolysis, and is also linked to lipid metabolism through the alternation of glycerol-3-phosphate and the pentose cycle. TIM is one of the most studied enzymes with more than 199 structures deposited in the PDB. The interest for this enzyme stems from the fact that it is involved in glycolysis, but also in aging, human diseases and metabolism. TIM has been a target in the search for chemical compounds against infectious diseases and is a model to study catalytic features. Until February 2017, 62% of all residues of the protein have been studied by mutagenesis and/or using other approaches. Here, we present a detailed and comprehensive recompilation of the reported effects on TIM catalysis, stability, druggability and human disease produced by each of the amino acids studied, contributing to a better understanding of the properties of this fundamental protein. The information reviewed here shows that the role of the noncatalytic residues depend on their molecular context, the delicate balance between the short and long-range interactions in concerted action determining the properties of the protein. Each protein should be regarded as a unique entity that has evolved to be functional in the organism to which it belongs. Proteins 2017; 85:1190-1211. © 2017 Wiley Periodicals, Inc.