Biomedical subjects

E Freire

Publications and source records attributed to E Freire.

At least 55 records · Page 3Linked to original sources

PubMed1997

Structure-based thermodynamic analysis of HIV-1 protease inhibitors.

A structural parametrization of the binding and folding energetics previously developed in this laboratory accounts quantitatively for the binding of 13 HIV-1 protease inhibitors for which high-resolution structures are available (A77003, A78791, A76928, A74704, A76889, VX478, SB203386, SB203238, SB206343, U100313, U89360, A98881, CGP53820). The binding free energies for the inhibitors are predicted with a standard deviation of +/- 1.1 kcal/mol or +/- 10%. Furthermore, the formalism correctly predicts the observed change in inhibition constant for the complex of A77003 and the resistant protease mutant V82A, for which the high-resolution structure is also available. The analysis presented here provides a structural mapping of the different contributions to the binding energetics. Comparison of the binding map with the residue stability map indicates that the binding pocket in the protease molecule has a dual character: half of the binding site is defined by the most stable region of the protein, while the other half is unstructured prior to inhibitor or substrate binding. This characteristic of the binding site accentuates cooperative effects that permit mutations in distal residues to have a significant effect on binding affinity. These results permit an initial assessment of the effects of mutations on the activity of protease inhibitors.

Binding Sites↗

PubMed1997

Structure-based statistical thermodynamic analysis of T4 lysozyme mutants: structural mapping of cooperative interactions.

The recent development of a structural parameterization of the energetics of protein folding has permitted the incorporation of the functions that describe the enthalpy, entropy and heat capacity changes, i.e. the individual components of the Gibbs energy, into a statistical thermodynamic formalism that describes the distribution of conformational states under equilibrium conditions. The goal of this approach is to construct with the computer a large ensemble of conformational states, and then to derive the most probable population distribution, i.e. the distribution of states that best accounts for a wide array of experimental observables. This analysis has been applied to four different mutants of T4 lysozyme (S44A, S44G, V131A, V131G). It is shown that the structural parameterization predicts well the stability of the protein and the effects of the mutations. The entire set of folding constants per residue has been calculated for the four mutants. In all cases, the effect of the mutations propagates beyond the mutation site itself through sequence and three-dimensional space. This phenomenon occurs despite the fact that the mutations are at solvent-exposed locations and do not directly affect other interactions in the protein. These results suggest that single amino acid mutations at solvent-exposed locations, or other locations that cause a minimal perturbation, can be used to identify the extent of cooperative interactions. The magnitude and extent of these effects and the accuracy of the algorithm can be tested by means of NMR-detected hydrogen exchange.

Bacteriophage T4↗

PubMed1997

Predicting the equilibrium protein folding pathway: structure-based analysis of staphylococcal nuclease.

The equilibrium folding pathway of staphylococcal nuclease (SNase) has been approximated using a statistical thermodynamic formalism that utilizes the high-resolution structure of the native state as a template to generate a large ensemble of partially folded states. Close to 400,000 different states ranging from the native to the completely unfolded states were included in the analysis. The probability of each state was estimated using an empirical structural parametrization of the folding energetics. It is shown that this formalism predicts accurately the stability of the protein, the cooperativity of the folding/unfolding transition observed by differential scanning calorimetry (DSC) or urea denaturation and the thermodynamic parameters for unfolding. More importantly, this formalism provides a quantitative account of the experimental hydrogen exchange protection factors measured under native conditions for SNase. These results suggest that the computer-generated distribution of states approximates well the ensemble of conformations existing in solution. Furthermore, this formalism represents the first model capable of quantitatively predicting within a unified framework the probability distribution of states seen under native conditions and its change upon unfolding.

Enzyme Stability↗

PubMed1996

Structure-based thermodynamic scale of alpha-helix propensities in amino acids.

A structural parameterization of the folding energetics has been used to predict the effect of single amino acid mutations at exposed locations in alpha-helices. The results have been used to derive a structure-based thermodynamic scale of alpha-helix propensities for amino acids. The structure-based thermodynamic analysis was performed for four different systems for which structural and experimental thermodynamic data are available: T4 lysozyme [Blaber et al (1994) J. Mol. Biol.235, 600-624], barnase [Horovitz et al. (1992) J.Mol.Biol.227,560-568], a synthetic leucine zipper [O'Neil & Degrado (1990) Science 250, 646-651], and a synthetic peptide [Lyu et al. (1990) Science 250, 669-673]. These studies have permitted the optimization of the set of solvent-accessible surface areas (ASA) for all amino acids in the unfolded state. It is shown that a single set of structure/thermodynamic parameters accounts well for all the experimental data sets of helix propensities. For T4 lysozyme, the average value of the absolute difference between predicted and experimental delta G values is 0.09 kcal/mol, for barnase 0.14 kcal/mol, for the synthetic coiled-coil 0.11 kcal/mol, and for the synthetic peptide 0.08 kcal/mol. In addition, this approach predicts well the overall stability of the proteins and rationalizes the differences in alpha-helix propensities between amino acids. The excellent agreement observed between predicted and experimental delta G values for all amino acids validates the use of this structural parameterization in free energy calculations for folding or binding.

Amino Acids↗

PubMed1996

Structure-based calculation of the equilibrium folding pathway of proteins. Correlation with hydrogen exchange protection factors.

A new statistical thermodynamic formalism has been developed in order to describe the equilibrium folding pathway of proteins. The resulting formalism allows calculation of the probabilities that individual amino acid residues will be in a native or native-like conformation for any given degree of folding of the protein molecule. The residue probabilities are defined by the probability distribution of conformational states and can be used to calculate experimental quantities like native-state, hydrogen exchange protection factors. A combinatorial algorithm aimed at generating a large ensemble of conformational states (10(4) to 10(6)) using the native structure as a template has been developed. The Gibbs energy and corresponding probability of each conformational state is estimated by using a previously developed structural parametrization of the energetics. The approach has been applied to five different proteins: hen egg-white lysozyme, equine lysozyme, bovine pancreatic trypsin inhibitor, staphylococcal nuclease and turkey ovomucoid third domain. The validity of the approach has been tested by comparing predicted and experimental hydrogen exchange protection factors. It is shown that for the above proteins 76%, 73%, 74%, 78% and 81% of all observed protection factors are predicted correctly. Furthermore, on average, the magnitude of the predicted protection factors, expressed as apparent free energies per residue deviate less than 1 kcal/mol from those obtained experimentally. These results represent the first attempt at predicting both the location and magnitude of hydrogen exchange protection factors from the high-resolution structure of a protein. The good agreement between experimental and predicted values has permitted a close examination of the nature of the equilibrium folding intermediates existing under conditions of maximal stability of the native state.

Animals↗

PubMed1996

Oxygen binding to fallow-deer (Dama dama) hemoglobin: stepwise enthalpies at pH 7.4.

High-precision thin-layer gas-solution microcalorimetry has been used to study the oxygen binding properties of fallow-deer (Dama dama) hemoglobin under physiological conditions. This method measures directly the enthalpy of macromolecular ligand binding by changing the ligand activity in a manner analogous to that of the Gill thin-layer optical apparatus ([1], D. Dolman and S. J. Gill, Anal. Biochem., 87 (1978) 127-134). By logarithmically lowering the partial pressure of oxygen we have generated differential heat binding curves of oxygen binding to fallow-deer hemoglobin in phosphate buffer at pH 7.4. In order to enlarge the data field, the temperature dependence of the oxygen affinity was examined by generating binding curves at a number of different temperatures allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumption of optical linearity between the various heme groups and reveals initially that overall oxygen binding to fallow-deer hemoglobin is less exothermic and of lower affinity than for human hemoglobin A0. In addition, previous optical work on the ancestrally related reindeer hemoglobin (Rangifer tarandus; [2], B. Giardina, O. Brix, M. Nuutinen, S. Sherbini, A. Bardgard, G. Lazzarino and S. Condo, FEBS Lett., 247 (1989) 135) has indicated that the enthalpy associated with its final two oxygen binding steps is minimal. Our calorimetric determination with fallow-deer hemoglobin also reveals this tendency. Presumably, this adaptation would make it easier for these animals to maintain a consistent hemoglobin oxygen saturation level under environmental conditions where the temperature fluctuates.

Animals↗

PubMed1996

Thermodynamic studies of the core histones: pH and ionic strength effects on the stability of the (H3-H4)/(H3-H4)2 system.

The self-associative behavior and the thermal stability of the H3/H4 histone complex was studied in low-ionic strength conditions by several physicochemical techniques, including differential scanning calorimetry and circular dichroism spectroscopy. At neutrality, the major molecular species present in solution is the (H3-H4)2 tetramer. Its thermodynamic properties cannot be studied directly though, since its thermal denaturation is completely irreversible even at the lowest salt concentrations. However, a complete thermodynamic analysis can be performed at low ionic strength and pH 4.5, where the (H3-H4)2 tetramer is quantitatively dissociated into two H3-H4 dimers and where almost complete reversibility of the thermal transitions is attained. The unfolding transition temperature of the 26.5 kDa H3-H4 dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. The thermal denaturation of the H3-H4 dimer is characterized by the presence of a single calorimetric peak, centered at 58 degrees C, with a corresponding enthalpy change of 25 kcal/mol of a 13 kDa monomer unit and a change in heat capacity upon unfolding of about 0.6 kcal/(K mol of 13 kDa monomer unit). The complex between histones H3 and H4 (tetramer or dimer) is stable between pH 9.5 and 3.0. At pH 1.5, the system is almost completely unfolded at all temperatures. At low ionic strengths and pH values between 5.0 and 2.5, the H3-H4 dimer behaves as a highly cooperative system, melting as a single unit; i.e. individual H3 and H4 folded monomers are not detectable during the treatment. The two-state mechanism accounting for the unfolding of the H3-H4 dimer at pH 4.5 is the same as that described for the H2A-H2B dimer at neutrality. Just like for the H2A and H2B histones, the H3 and H4 polypeptides are properly folded only when assembled as H3-H4 dimers or in higher-order histone assemblies. Therefore, coupling along the interfaces of the two chains within the heterodimer is the major factor contributing to the stabilization of the secondary and tertiary structures of the chains as well as of the histone dimers.

Animals↗

PubMed1996

The magnitude of the backbone conformational entropy change in protein folding.

The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent-exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (delta Cp), identical solvent-related entropies of unfolding (delta Ssolv), and identical enthalpies of unfolding (delta H) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles phi and phi. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 +/- 0.2 cal/K. mol with respect to that of glycine, consistent with a 3.4-fold reduction in the number of allowed conformations in the alanine-containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position beta. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K. mol in the backbone entropy was obtained for leucine and lysine, respectively. beta-branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K.mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an alpha-helix is 6.51 cal/K.mol for glycine. These and previous results have allowed a complete estimation of the conformational entropy changes associated with protein folding.

Alanine↗

PubMed1996

The enthalpy change in protein folding and binding: refinement of parameters for structure-based calculations.

Two effects are mainly responsible for the observed enthalpy change in protein unfolding: the disruption of internal interactions within the protein molecule (van der Waals, hydrogen bonds, etc.) and the hydration of the groups that are buried in the native state and become exposed to the solvent on unfolding. In the traditional thermodynamic analysis, the effects of hydration have usually been evaluated using the thermodynamic data for the transfer of small model compounds from the gas phase to water. The contribution of internal interactions, on the other hand, are usually estimated by subtracting the hydration effects from the experimental enthalpy of unfolding. The main drawback of this approach is that the enthalpic contributions of hydration, and those due to the disruption of internal interactions, are more than one order of magnitude larger than the experimental enthalpy value. The enthalpy contributions of hydration and disruption of internal interactions have opposite signs and cancel each other almost completely resulting in a final value that is over 10 times smaller than the individual terms. For this reason, the classical approach cannot be used to accurately predict unfolding enthalpies from structure: any error in the estimation of the hydration enthalpy will be amplified by a factor of 10 or more in the estimation of the unfolding enthalpy. Recently, it has been shown that simple parametric equations that relate the enthalpy change with certain structural parameters, especially changes in solvent accessible surface areas have considerable predictive power. In this paper, we provide a physical foundation to that parametrization and in the process we present a system of equations that explicitly includes the enthalpic effects of the packing density between the different atoms within the protein molecule. Using this approach, the error in the prediction of folding/unfolding enthalpies at 60 degrees C, the median temperature for thermal unfolding, is better than +/- 3% (standard deviation = 4 kcal/mol).

Protein Conformation↗

PubMed1995

Precise scanning calorimeter for studying thermal properties of biological macromolecules in dilute solution.

A precise scanning calorimeter for studying the heat capacity of liquids in a broad temperature range has been developed. By its design and capabilities this calorimeter is the first of a new generation for this type of instrument. This new scanning calorimeter operates differentially, is equipped with a pair of gold capillary cells and semiconductor sensors, and is able to scan up and down in temperature at user-selected rates. This instrument is completely operated by an integrated computer which also provides a full thermodynamic analysis of the results. Its construction does not involve the use of organic compounds, thus eliminating a source of baseline noise that has affected previous calorimeters. The operational temperature range of the instrument can be varied between 0 and 120 degrees C. The gold capillary cells (operational volume 0.8 ml) minimize temperature gradients in the heated/cooled liquid sample and permit easy washing and reloading without air bubbles. These features are crucial for the accuracy of difference heat capacity measurements and determination of the absolute value of the partial heat capacity of solute molecules. The measurements can be performed under an excess constant pressure (up to 3 atm) to prevent formation of gas bubbles and boiling of aqueous solutions above 100 degrees C. The noise level of the recorded heating/cooling power difference is below 50 x 10(-9) W (i.e., below 10 ncal/s) with a response half-time of 5 s. The reproducibility of the baseline without refilling the capillary cells is on the order of 0.5 x 10(-6) W. Reproducibility of the baseline upon refilling the cell is of the same order of magnitude. This provides an accuracy in difference heat capacity determination on the order of 10 mu cal/degrees Kml at a heating rate of 1 degree K/min.

Calorimetry, Differential Scanning↗

PubMed1995

Subunit III of cytochrome c oxidase influences the conformation of subunits I and II: an infrared study.

The secondary structure of wild-type Paracoccus denitrificans cytochrome c oxidase obtained by decomposition of the infrared amide I band contains 44% alpha-helix, 18% beta-sheet, 14% beta-turns, 18% loops, and 6% nonordered segments. The mutant lacking subunit III presents a small but significant increase (from 18% to 24%) in the percentage of loops and slight differences in the other components. Using band/area ratios and tyrosine side chain absorption as an inner standard, it is shown that in the absence of subunit III the structure of subunits I and II is altered although no changes in their alpha-helix or beta-sheet content are observed. In the bacterial oxidase, thermal infrared studies show a complex denaturation pattern characterized by the presence of a partially denatured intermediate state. Of the seven predicted subunit III alpha-helices, only four are resistant toward the thermal challenge and behave as expected for typical transmembrane helices. The observation that the absence of subunit III influences the conformation of loop regions in the two other subunits suggests that part of the interaction surface between subunit III and the catalytic subunits might be located outside the lipid bilayer.

Electron Transport Complex IV↗

PubMed1995

The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme.

The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and alpha-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the alpha and beta domains. In equine lysozyme and alpha-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the alpha and beta domains unfold as a single cooperative unit, in equine lysozyme the two domains unfold in two separate cooperative stages even in the presence of excess Ca2+. The calcium binding beta-domain unfolds at a lower temperature and with more extensive heat absorption than the alpha-domain. Binding of Ca2+ increases the stability of the beta-domain, but even in the holo form it is less stable than the alpha-domain. The thermodynamic characteristics of Ca2+ binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of alpha-lactalbumin, which also unfolds in two stages, but in the latter case the second stage is much less cooperative and proceeds with a smaller and diffuse heat absorption. As a result, the total enthalpy of unfolding of equine lysozyme is significantly larger than that of alpha-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-state transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in equine lysozyme is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the alpha and beta domains.

Animals↗

PubMed1995

Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin.

The discovery that the protease from the human immunodeficiency virus (HIV) belongs to the aspartic protease family has generated renewed interest in this class of proteins. In this paper, the interactions of endothiapepsin, an aspartic proteinase from the fungus Endothia parasitica, with the inhibitor pepstatin A have been studied by high-sensitivity calorimetric techniques. These experiments have permitted a complete characterization of the temperature and pH-dependence of the binding energetics. The binding reaction is characterized by negative intrinsic binding enthalpy and negative heat capacity changes. The association constant is maximal at low pH (2 x 10(9) M-1 at pH 3) but decreases upon increasing pH (8.1 x 10(6) M-1 at pH 7). The binding of the inhibitor is coupled to the protonation of one of the aspartic moieties in the Asp dyad of the catalytic site of the protein. This phenomenon is responsible for the decrease in the apparent affinity of the inhibitor for the enzyme upon increasing pH. The experimental results presented here indicate that the binding of the inhibitor is favored both enthalpically and entropically. While the favorable enthalpic contribution is intuitively expected, the favorable entropic contribution is due to the large gain in solvent-related entropy associated with the burial of a large hydrophobic surface, that overcompensates the loss in conformational and translational/rotational degrees of freedom upon complex formation. The characteristics of the molecular recognition process have been evaluated by means of structure-based thermodynamic analysis. Three regions in the protein contribute significantly to the free energy of binding: the residues surrounding the Asp dyad (Asp32 in the N-terminal lobe and Asp215 in the C-terminal domain) and the flap region (Ile73 to Asp77). In addition, the rearrangement of residues that are not in immediate contact with the inhibitor provides close to 40% of the protease contribution to the binding free energy. On the other hand, the two statine residues provide more than half of the inhibitor contributions to the total free energy of binding. It is demonstrated that a previously developed empirical structural parametrization of the thermodynamic parameters that define the Gibbs energy, accurately accounts for the binding energetics and its temperature and pH-dependence.

Aspartic Acid Endopeptidases↗

PubMed1995

Forces and factors that contribute to the structural stability of membrane proteins.

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.

Amino Acid Sequence↗

PubMed1995

Thermodynamic studies of the core histones: ionic strength and pH dependence of H2A-H2B dimer stability.

The thermal stability of the core histone dimer H2A-H2B has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The unfolding transition temperature of the 28 kDa H2A-H2B dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. At neutral pH and physiological ionic strength, the thermal denaturation is centered at about 50 degrees C with a corresponding enthalpy change of about 40 kcal/mol of 14 kDa monomer unit and an excess heat capacity of about 1.4 kcal/(K.mol) of 14 kDa monomer unit. The H2A-H2B dimer is stable mainly between pH 5.5 and 10.5. Below pH 4.0, the system is unfolded at all temperatures. The thermodynamic analysis is performed at low ionic strength where almost complete reversibility is attained, since higher salt conditions seem to promote aggregation and irreversibility of the transitions. Analysis of the data shows that at low ionic strength and pH values between 6.5 and 8.5, the H2A-H2B dimer behaves as a highly cooperative system, melting as a single unit without any detectable intermediates of dissociated, yet folded, H2A and H2B monomers. This is consistent with the observed protein concentration dependence of the midpoint of the thermal denaturation. The two-state unfolding process can be described by the general scheme AB-->2U, indicating that the individual H2A and H2B polypeptides are folded, stable entities only when complexed as the H2A-H2B dimer and that the major contribution to the stabilization of the dimer derives from the coupling between the H2A and H2B interfaces.

Animals↗

PubMed1995

Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor.

The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N4<==>4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 microM have a transition temperature (Tm) of 75.3 degrees C while at 0.5 microM the value drops to 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)-1 with a heat capacity change of 387 cal (K.mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

PubMed1995

Forces and factors that contribute to the structural stability of membrane proteins.

Amino Acid Sequence↗

PubMed1995

GroES and the chaperonin-assisted protein folding cycle: GroES has no affinity for nucleotides.

The E. coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins. GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold. On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested [Martin, et al., Nature, 366 (1993) 279-282]. We confirm the photolabeling of GroES with 8-azido-ATP. However, other proteins not known to contain nucleotide binding sites also became photolabeled suggesting that labeling is non-specific. Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected. We conclude that GroES has no nucleotide binding site.

Adenosine Triphosphate↗