Yeast Phosphoglycerate Kinase Research Articles

Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo.

Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.

Site-directed mutagenesis of yeast phosphoglycerate kinase: Arginines 65, 121 and 168

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). K m values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured K m's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the K m's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher k cat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg 121 and Arg 121 with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.

Interaction of ATP analogs with yeast 3-phosphoglycerate kinase. Affinity labeling of the hinge region.

Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.

Arrangement of substrates at the active site of yeast phosphoglycerate kinase. Effect of sulfate ion.

beta, gamma-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a probe of the conformation of the substrates at the active site of yeast phosphoglycerate kinase (PGK). The paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the 1H, 31P, and 13C nuclei of 3-phospho-D-glycerate (3PGA) were examined to determine the distances between enzyme-bound CrATP and the nuclei of 3PGA in the ternary PGK.CrATP.3PGA complex. Kinetic studies indicated that Ki(CrATP) was dependent on the concentration of 3PGA that was abolished in the presence of activator sulfate ion. Similarly, NMR studies showed that the environment of CrATP bound at the active site was altered by the presence of sulfate ion. The determined metal-to-nucleus distances show the close proximity of the substrates at the active site of PGK, which is consistent with a hinge-bending mechanism of the enzyme for bringing the two substrates together. Additionally, the presence of an activating concentration of sulfate was observed to cause significant changes in both the relative Cr(3+)-to-3PGA distances and the overall conformation of 3PGA when compared to the findings in the absence of sulfate. This is consistent with the active site being in a "less closed" configuration when sulfate is present. Under both conditions, models can be constructed that allow for direct transfer of a phosphoryl group between the bound substrates.

Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process.

The unfolding-refolding kinetics of yeast phosphoglycerate kinase were studied using the chemical reactivity of genetically introduced cysteinyl residues as conformational probes and far-ultraviolet circular dichroism. A unique internal cysteinyl residue was introduced in several mutants at selected positions in the N- and C-domains. The cysteinyl residues were at positions 97 (the unique cysteinyl residue of the wild-type enzyme), 183 in the N-domain, 285 and 324 in the C-domain. A similar strategy has been used to study the unfolding-refolding transition under equilibrium conditions [Ballery et al. (1990) Protein Eng. 3, 199-204]. Except for the mutant C97A,A183C, whose cysteinyl residue is located at the domain interface, three labeling phases were observed during the refolding process, indicating the presence of three species, the unfolded, intermediate, and folded proteins. The comparison of the data obtained following the accessibility of the thiol group to 5,5'-dithiobis(2-nitrobenzoate) and ellipticity at 218 nm indicated that all mutants have the same folding pathway and allowed us to characterize the intermediate. In this species, each domain appeared to have a high content of secondary structure but a flexible tertiary structure; this intermediate, which had the characteristics of a molten globule, remained in fluctuating equilibrium with a widely unfolded form. The same folding intermediate was detected for mutant C97A,A183C; however, the cysteinyl residue being totally accessible to the reagent, it is likely that in this intermediate the interdomain interactions are not established. Domain pairing and formation of the native tertiary structure occur simultaneously in the slow phase of refolding. The validity and limitations of the methodology are discussed.

Microcalorimetry of enzyme–substrate binding: yeast phosphoglycerate kinase

The binding of substrates and various other anions to yeast phosphoglycerate kinase (PGK) has been studied by titration microcalorimetry. Binding of 3-phosphoglycerate to a single high-affinity site (K≈ 2 × 105 dm3 mol–1) is entropy driven (ΔG0≈–30 kJ mol–1, ΔH0≈–6 kJ mol–1, ΔS0≈+80 J K–1 mol–1), with additional heat effects arising from deprotonation of the substrate near neutral pH. This binding is inhibited by a range of other anions (chloride, sulfate, triphosphate, other substrates), probably through competition for the same basic site. Nucleotide substrates (ATP and ADP) appear to bind to two separate sites on the enzyme, one of which is competitive with the phosphoglycerate site and with other anions. Microcalorimetry of non-productive ternary complex for mation (PGK–ADP–3-PG) reflects this multiplicity of sites. Comparative experiments with two site-directed mutants of PGK (His-388 → Gln and Arg-168 → Lys) are also reported.

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site

Exchange-inert β,γ-bidentate Cr(H 2O) x(NH 3) yATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min −1 to 0.118 min −1 for Cr(NH 3) 4ATP and Cr(H 2O) 4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k obs by 3-PGA was proportional to the number of NH 3 ligands present in the coordination sphere of Cr 3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr 3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H 2O) 4ATP. Δ diastereomer of Cr(H 2O) 4ATP yielded an order of magnitude smaller K i value compared to the value observed with the Λ isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H 2O) 4ATP or Cr(NH 3) 4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr 3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme ·CrATP and enzyme·CrATP·3-PGA complexes.

The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae.

A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell-free extracts. The system was used to examine the influence of 5'-capping, 3'-polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat-shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap-independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

Insertion of an 18-nucleotide-long poly(G) tract into the 3'-terminal untranslated region of yeast phosphoglycerate kinase (PGK1) mRNA increases its chemical half-life by about a factor of 2 (P. Vreken, R. Van der Veen, V. C. H. F. de Regt, A. L. de Maat, R. J. Planta, and H. A. Raué, Biochimie 73:729-737, 1991). In this report, we show that this insertion also causes the accumulation of a degradation intermediate extending from the poly(G) sequence down to the transcription termination site. Reverse transcription and S1 nuclease mapping experiments demonstrated that this intermediate is the product of shorter-lived primary fragments resulting from endonucleolytic cleavage immediately downstream from the U residue of either of two 5'-GGUG-3' sequences present between positions 1100 and 1200 close to the 3' terminus (position 1251) of the coding sequence. Similar endonucleolytic cleavages appear to initiate degradation of wild-type PGK1 mRNA. Insertion of a poly(G) tract just upstream from the AUG start codon resulted in the accumulation of a 5'-terminal degradation intermediate extending from the insertion to the 1100-1200 region. RNase H degradation in the presence of oligo(dT) demonstrated that the wild-type and mutant PGK1 mRNAs are deadenylated prior to endonucleolytic cleavage and that the half-life of the poly(A) tail is three- to sixfold lower than that of the remainder of the mRNA. Thus, the endonucleolytic cleavage constitutes the rate-limiting step in degradation of both wild-type and mutant PGK1 transcripts, and the resulting fragments are degraded by a 5'----3' exonuclease, which appears to be severely retarded by a poly(G) sequence.

The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis

The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min −1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min −1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

Insertion of an 18-nucleotide-long poly(G) tract into the 3'-terminal untranslated region of yeast phosphoglycerate kinase (PGK1) mRNA increases its chemical half-life by about a factor of 2 (P. Vreken, R. Van der Veen, V. C. H. F. de Regt, A. L. de Maat, R. J. Planta, and H. A. Raué, Biochimie 73:729-737, 1991). In this report, we show that this insertion also causes the accumulation of a degradation intermediate extending from the poly(G) sequence down to the transcription termination site. Reverse transcription and S1 nuclease mapping experiments demonstrated that this intermediate is the product of shorter-lived primary fragments resulting from endonucleolytic cleavage immediately downstream from the U residue of either of two 5'-GGUG-3' sequences present between positions 1100 and 1200 close to the 3' terminus (position 1251) of the coding sequence. Similar endonucleolytic cleavages appear to initiate degradation of wild-type PGK1 mRNA. Insertion of a poly(G) tract just upstream from the AUG start codon resulted in the accumulation of a 5'-terminal degradation intermediate extending from the insertion to the 1100-1200 region. RNase H degradation in the presence of oligo(dT) demonstrated that the wild-type and mutant PGK1 mRNAs are deadenylated prior to endonucleolytic cleavage and that the half-life of the poly(A) tail is three- to sixfold lower than that of the remainder of the mRNA. Thus, the endonucleolytic cleavage constitutes the rate-limiting step in degradation of both wild-type and mutant PGK1 transcripts, and the resulting fragments are degraded by a 5'----3' exonuclease, which appears to be severely retarded by a poly(G) sequence.

Characterization of the structure and properties of the His 62-->Ala and Arg 38-->Ala mutants of yeast phosphoglycerate kinase: an investigation of the catalytic and activatory sites by site-directed mutagenesis and NMR.

The role of two "basic patch" residues, Arg-38 and His-62, in the catalytic function and anion-dependent activation of yeast 3-phosphoglycerate kinase (PGK) was investigated by site-directed mutagenesis. Steady-state kinetics and NMR experiments were conducted to characterize the functional properties and structural integrity of the R38A and H62A mutants. The results of these studies, in combination with earlier mutagenesis experiments, suggest that Arg-38 is the only catalytically essential residue among the conserved histidines and arginines of the basic patch. It appears that, similar to the remaining basic patch residues, His-62 is important for anion-dependent activation but not for enzyme activity. Cumulative evidence from this study and from previous mutagenesis experiments suggests that the basic patch region is in effect an extended anion binding site that encompasses both the catalytic and the general anion-binding site. It is proposed that substitution of any one of the basic patch residues results in an increased localization of the catalytic site. Substrate and product may still bind to this site, but a simultaneous binding of activatory anions, required for activation, has been impaired. NMR experiments suggest that the conformational changes observed upon binding of 3-PG to wild-type PGK are necessary for anion- and substrate-dependent activation.

Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins.

The dependence of the fluorescence emission maximum of the tryptophan residues in several two-tryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A2) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case of metalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.

An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation.

This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H-NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3-phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5-triphosphate, 4-amino-6-trichloroethenyl-1,3- benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported. Evidence that binding to the general anion site in the 'basic patch' region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.

Translational efficiency of mRNA in yeast cells is not increased by plant viral leader sequences

Synthetic oligonucleotides encoding the 5′-non-translated (leader) sequence of the coat protein mRNA of alfalfa mosaic virus RNA 4 or the Ω′ leader sequence of tobacco mosaic virus RNA were used to replace the natural leader region of the yeast phosphoglycerate kinase (PGK1) mRNAs and the translational efficiency of the chimeric mRNA was determined in yeast cells. In neither case did we observed a significant increase compared to the translational efficiency shown by the wild-type PGK mRNA, in contrast to the known stimulatory effect of these leader sequences on translation in mammalian, plant and bacterial in-vivo and/or in-vitro systems. The same result was obtained when the translational efficiencies in yeast cells of Escherichia coli β-galactosidase mRNAs carrying the PGK or either of the two viral leader sequences were compared.

Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.

Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.

Thermodynamics of Structural Stability and Cooperative Folding Behavior in Proteins

Publisher Summary This chapter provides an insight that quantitative predictions of the stability and cooperative folding behavior of proteins are within reach. The problem of predicting the structure of a protein from its amino acid sequence constitutes the classical “protein folding problem.” The thermodynamic aspects of this problem constitute the focus of the chapter. Experimental thermodynamic values for the fundamental forces that determine protein stability are refined to a point in which accurate calculations of the free energy of stabilization are possible, provided that the protein structure is known. The chapter also (1) predicts the overall stability of proteins based on structural information, (2) predicts the cooperative folding/unfolding behavior of proteins, (3) predicts the magnitude of the interactions among structural domains in proteins, and (4) predicts the presence of specific partially folded intermediates. Estimation of the folding/unfolding partition function using a hierarchical approximation and subsequent prediction of cooperative behavior is also increasingly feasible, as demonstrated for myoglobin and yeast phosphoglycerate kinase. Thermodynamic stability and cooperative interactions in proteins can be determined by hierarchical approach to partition functions, thermodynamic dissection of cooperative interactions, and classification of cooperative forces and mechanisms. The strategy used to dissect specific fundamental interactions is to isolate first the hydrophobic effects and subsequently other interactions. Some future work will extend the capability to proteins exhibiting other than pure α-helical structural motifs.

Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through hydrophobic cluster analysis and molecular modelling.

A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain. In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken. We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK). This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method. According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides. Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase. These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane. Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.

A cell-free extract from yeast cells for studying mRNA turnover.

We have isolated a cell-free extract from yeast cells that reproduces the differences observed in vivo in the rate of turnover of individual yeast mRNAs. Detailed analysis of the degradation of yeast phosphoglycerate kinase (PGK) mRNA in this system demonstrated that both natural and synthetically prepared PGK transcripts are degraded by the same pathway previously established by us in vivo, consisting of endonucleolytic cleavage at a number of 5'-GGUG-3' sequence motifs within a short target region located close to the 3'-end of the coding sequence followed by 5'-3' exonucleolytic removal of the resulting fragments. The extract, therefore, is suitable for studying the mechanistic details of mRNA turnover in yeast. As a first application of this system we have performed a limited mutational analysis of two of the GGUG motifs within the endonucleolytic target region of the PGK transcript. The results show that sequence changes in either motif abolish cleavage at the mutated site only, indicating the involvement of the residues in question in selection of the cleavage positions.