Chaperonin-affected folding of globular proteins.

We studied the effect of GroEL on the kinetic refolding ofα-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-γS and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.

Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches.

The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results.

Nucleotide-induced transition of GroEL from the high-affinity to the low-affinity state for a target protein: effects of ATP and ADP on the GroEL-affected refolding of alpha-lactalbumin.

We studied the refolding kinetics of alpha-lactalbumin in the presence of wild-type GroEL and its ATPase-deficient mutant D398A at various concentrations of nucleotides (ATP and ADP). We evaluated the apparent binding constant between GroEL and the alpha-lactalbumin refolding intermediate quantitatively by numerical simulation analysis of the alpha-lactalbumin refolding curves in the presence and absence of GroEL. The binding constant showed a co-operative decrease with an increase in ATP concentration, whereas the binding constant decreased in a non-co-operative manner with respect to ADP concentration. For the D398A mutant, the ATP-induced decrease in affinity occurred much faster than the steady-state ATP hydrolysis by this mutant, suggesting that ATP binding to GroEL rather than ATP hydrolysis, was responsible for the co-operative decrease in the affinity for the target protein. We thus analyzed the nucleotide-concentration dependence of affinity of GroEL for the target protein using an allosteric Monod-Wyman-Changeux model in which GroEL underwent an ATP-induced co-operative conformational transition between the high-affinity and low-affinity states of the target protein. The transition midpoint of the ATP-induced transition of GroEL has been found to be around 30 microM, in good agreement with the midpoint evaluated in other structural studies of GroEL. The results show that the observed difference between ATP and ADP-induced transitions of GroEL are brought about by a small difference in an allosteric parameter (the ratio of the nucleotide affinities of GroEL in the high-affinity and the low-affinity states), i.e. 4.1 for ATP and 2.6 for ADP.

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP.

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.

[A case of bilateral coronal craniosynostosis with the P250R mutation in FGFR3 gene].

Recently, the substitution of proline 250 by arginine in the fibroblast growth factor receptor 3 (FGFR3) gene, has been identified in patients with craniosynostosis and defines a new syndrome on a molecular basis. We report a 1-year-1-month-old female with bilateral coronal craniosynostosis who had the P250R mutation in FGFR3 gene detected by DNA sequencing. She had brachycephaly, temporal bossing, high and flat forehead, hypertelorism, mild proptosis, low set ears and no digital abnormalities. She underwent surgical repair at 7 months and her cosmetic problems were improved. Her development was normal up to 13 months of age. DNA analysis from her parents showed that her father had the same mutation. The phenotypes of the P250R mutation in the FGFR3 syndrome are variable even within the same family, but main characteristic clinical features are follows, 1) lateral or bilateral coronal craniosynostosis, 2) mild hand and foot anomalies, and 3) sensory deafness. In FGFR3 syndrome the diagnosis of P250R mutation by polymerase chain reaction (PCR) is very easy and important for early diagnosis and genetic counseling.

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations.

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy.

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.

Folding of green fluorescent protein and the cycle3 mutant.

Although the correct folding of green fluorescent protein (GFP) is required for formation of the chromophore, it is known that wild-type GFP cannot mature efficiently in vivo in Escherichia coli at 37 degrees C or higher temperatures that the jellyfish in the Pacific Northwest have never experienced. Recently, by random mutagenesis by the polymerase chain reaction (PCR) method, a mutant called Cycle3 was constructed. This mutant had three mutations, F99S, M153T, and V163A, on or near the surface of the GFP molecule and was able to mature correctly even at 37 degrees C [Crameri et al. (1996) Nat. Biotechnol. 143, 315-319]. In the present study, we investigated the differences in their folding behavior in vitro. We observed the folding and unfolding reactions of both wild-type GFP and the Cycle3 mutant by using green fluorescence as an indicator of the formation of the native structure and examining hydrogen-exchange reactions by Fourier transform infrared spectroscopy. Both proteins showed unusually slow refolding and unfolding rates, and their refolding rates were almost identical under the native state at 25 and at 35 degrees C. On the other hand, aggregation studies in vitro showed that wild-type GFP had a strong tendency to aggregate, while the Cycle3 mutant did not. These results indicated that the ability to mature efficiently in vivo at 37 degrees C was not due to the improved folding and that reduced hydrophobicity on the surface of the Cycle3 mutant was a more critical factor for efficient maturation in vivo.

Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule.

The native state (1)H, (15)N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state.

Limitations of urinary telomerase activity measurement in urothelial cancer.

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10(6) cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37 degrees C and 25 degrees C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37 degrees C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25 degrees C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster.

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.

Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme.

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme.

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.

Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding.

The GroES binding site at the apical domain of GroEL, mostly consisting of hydrophobic residues, overlaps largely with the substrate polypeptide binding site. Essential contribution of hydrophobic interaction to the binding of both GroES and polypeptide was exemplified by the mutant GroEL(L237Q) which lost the ability to bind either of them. The binding site, however, contains three hydrophilic residues, E238, T261, and N265. For GroES binding, N265 is essential since GroEL(N265A) is unable to bind GroES. E238 contributes to rapid GroES binding to GroEL because GroEL(E238A) is extremely sluggish in GroES binding. Polypeptide binding was not impaired by any mutations of E238A, T261A, and N265A. Rather, these mutants, especially GroEL(N265A), showed stronger polypeptide binding affinity than wild-type GroEL. Thus, these hydrophilic residues have a dual role; they help GroES binding on one hand but attenuate polypeptide binding on the other hand.

Is folding of beta-lactoglobulin non-hierarchic? Intermediate with native-like beta-sheet and non-native alpha-helix.

The refolding of beta-lactoglobulin, a beta-barrel protein consisting of beta strands betaA-betaI and one major helix, is unusual because non-native alpha-helices are formed at the beginning of the process. We studied the refolding kinetics of bovine beta-lactoglobulin A at pH 3 using the stopped-flow circular dichroism and manual H/(2)H exchange pulse labeling coupled with heteronuclear NMR. The protection pattern from the H/(2)H exchange of the native state indicated the presence of a stable hydrophobic core consisting of betaF, betaG and betaH strands. The protection pattern of the kinetic intermediate obtained about one second after initiating the reaction was compared with that of the native state. In this relatively late kinetic intermediate, which still contains some non-native helical structure, the disulfide-bonded beta-hairpin made up of betaG and betaH strands was formed, but the rest of the molecule was fluctuating, where the non-native alpha-helices may reside. Subsequently, the core beta-sheet extends, accompanied by a further alpha-helix to beta-sheet transition. Thus, the refolding of beta-lactoglobulin exhibits two elements: the critical role of the core beta-sheet is consistent with the hierarchic mechanism, whereas the alpha-helix to beta-sheet transition suggests the non-hierarchic mechanism.

Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.

An AciI polymorphism in the 3' untranslated region of the human phosphomannomutase 2 (PMM2) gene.

We found an AciI polymorphism in the 3' untranslated region of the phosphomannomutase 2 (PMM2) gene located at 16p13. A G-to-C transition at nucleotide position 96 bp downstream from the PMM2 stop codon was detected in polymerase chain reaction (PCR) products after AciI digestion. The heterozygosity of the polymorphic alleles was 0.375 in a Japanese population. This polymorphism is useful for genetic analysis in patients with carbohydrate-deficient glycoprotein syndromes, of which there are four subtypes.

Missense mutations in the phosphomannomutase 2 gene of two Japanese siblings with carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein syndrome type I (CDG1) is an autosomal recessive disorder characterized by severe nervous system involvement and a carbohydrate moiety deficiency in N-linked glycoproteins. Clinical symptoms are psychomotor retardation, stroke-like episodes or hemorrhagic episodes, hepatic dysfunction, polyneuropathy, and cerebellar ataxia. Marked atrophy of the cerebellar hemispheres and pons is recognizable on CT scan or MRI. CDGI has been mapped to human chromosome 16p by linkage studies. Recently, missense mutations in the gene for phosphomannomutase (PMM2) have been detected in Caucasian patients with CDG1. We studied DNA mutations in PMM2 in a Japanese family with CDG1. DNA sequencing of PMM2 in the siblings showed missense mutations of maternal origin in exon 5 and of paternal origin in exon 8. No such mutations were detected in 50 unrelated healthy Japanese. These findings suggest that the PMM2 is responsible for CDG1 in the Japanese as well as in Caucasians, and CDG1 may be the diagnosis in OPCA of neonatal onset, more often than currently thought.