Role of an evolutionarily invariant serine for the stability of human carbonic anhydrase II.

There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp-209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29----Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O----S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group.

Folding around the C-terminus of human carbonic anhydrase II. Kinetic characterization by use of a chemically reactive SH-group introduced by protein engineering.

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH-group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C-terminal part of the polypeptide chain, which is involved in a unique 'knot' topology, was investigated. Our results show that the structure around the C-terminal, composed of the outermost beta-strands in a dominating beta-structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that beta-strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of beta-structure.

Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 9 min). Kinetic data from 'double-jump' measurements indicate that proline isomerization might be rate determining in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics: the presence of isomerase during reactivation lowers the half-time of the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated enzyme. Thus, cis-trans isomerization of the Pro202 cis-peptidyl bond is not rate determining in the reactivation process. The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

Melatonin together with noradrenaline augments contractions of human myometrium.

The hormone melatonin is known to influence the circadian rhythm, and it probably also mediates some of the physiological changes that occur in the body at night. Inasmuch as uterine activity is greater during darkness, we investigated whether melatonin could modulate uterine contractility. Biopsies were performed during caesarean sections to obtain uterine tissue from women who had reached full term. The obtained samples were mounted in organ baths, and spontaneous contractions were recorded. Melatonin alone did not change myometrial contractility, whereas melatonin in combination with noradrenaline potentiated contractions. These results may indicate that melatonin plays a role in the timing of labour, since labour often begins late in the evening.

Noradrenaline-induced pigment aggregating response of melanophores in normal, denervated and reinnervated cichlid skin.

The present study examines noradrenaline (NA) effects on melanophore pigment aggregation in normal, denervated and reinnervated teleost skin in vitro. Many axons were present in the melanophore-containing layer of normal skin. One week after a nerve crush lesion the skin was devoid of axons. By 1 month the skin was partly reinnervated. One day after nerve crush NA-sensitivity was markedly increased compared to controls. Sensitivity then approached normality but it remained elevated for at least one month. We conclude that melanophore supersensitivity develops very rapidly upon denervation and then gradually fades away during reinnervation.

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.

A melatonin binding site modulates the alpha 2-adrenoceptor.

The melanophores of the cuckoo wrasse (Labrus ossifagus L., a teleost fish) can aggregate and disperse their pigment granules. This migration is controlled by sympathetic nerves and a postsynaptic alpha 2-adrenoceptor. Melatonin was discovered because of its ability to aggregate pigment granules, hence we used the cuckoo wrasse melanophore as a model for studying the effect of melatonin at a cellular level. We found that melatonin had no aggregating effect; instead the hormone enhanced the actions of several alpha 2-agonists, such as noradrenaline, medetomedine and clonidine. When the melanophores were pre-aggregated in vitro by use of the alpha 2-agonist B-HT 920, the aggregation was not augmented after the addition of melatonin. Instead the pre-aggregated granules were dispersed. This suggests that melatonin has affinity for an alpha 2-adrenoceptor site that can modulate the effect of alpha 2-adrenoceptor agonists.

Is Ca2+ the second messenger in the response to melatonin in cuckoo wrasse melanophores?

Pigment aggregation in melanophores of Labrus ossifagus is controlled by an alpha2-adrenoceptor and is somehow modulated by melatonin. The signal transduction mechanisms seem to involve both an attenuation of cAMP and an increase in intracellular Ca2+, inhibiting protein kinase A or activating a phosphatase, respectively. These effects result in dephosphorylation, which in turn induces aggregation. Various alpha2-adrenoceptor agonists attenuate cAMP levels or increase the concentration of intracellular Ca2+. Noradrenaline, for example, lowers cAMP but does not affect the calcium signal whereas B-HT 920, an alpha2-adrenoceptor specific agonist, does not induce a cAMP decrease but does appear to induce an increase in intracellular Ca2+. This later inference is drawn from experiments with BAPTA/AM, an intracellular calcium chelator, which counteracts the aggregation induced by B-HT 920. Interestingly, the very potent alpha2-adrenoceptor agonist medetomidine apparently activates both signal transduction pathways, which could explain its high efficacy in producing aggregation. Melatonin itself does not cause pigment aggregation, but it potentiates noradrenaline-induced aggregation. It has been suggested that melatonin receptors and alpha2-adrenoceptors follow the same signal transduction pathway, i.e. an attenuation of cAMP. In our experiments, melatonin did not reduce cAMP levels; instead it appears to increase Ca2+ concentration, since melatonin-potentiated aggregation was inhibited by BAPTA/AM. Thus, aggregation amplified by melatonin is probably not mediated by a further decrease in cAMP, but by the same signal transduction mechanism as B-HT 920, i.e. an increase in Ca2+. This further strengthens the suggestion that melatonin and B-HT 920 bind to the same site, but it is unclear if that particular site is on the melatonin receptor or the alpha2-adrenoceptor.

Effect of wall tension on DNA and protein synthesis in bovine mesenteric arteries in vitro.

The aim of this investigation was to study the effect of wall tension and calcium antagonists on DNA and protein synthesis in bovine mesenteric arteries in vitro. The wall tension of the bovine mesenteric arteries was raised by stretching the vessel wall perpendicular to the length axis of the vessel. DNA and protein synthesis were determined by measuring incorporation of 3H-thymidine into DNA and incorporation of 14C-leucine into protein respectively. Elevating the wall tension from 0.05 N to 0.5 N significantly increased 3H-thymidine incorporation and 14C-leucine incorporation after an incubation period of 3 hr. Stretch had no effect on the distribution of 3H-thymidine. The distribution of 14C-leucine was increased by stretch in regular medium and to a less extent in calcium-free medium, which suggest that stretch stimulates the membrane transport of 14C-leucine. When the tension was increased from 0.05 N to 0.5 N for 10 min. before the incubation with 3H-thymidine, no effect was found. One microM nifedipine or felodipine inhibited the increase in 3H-thymidine incorporation caused by stretching, while no effect was found on 14C-leucine incorporation. In calcium-free medium, stretch-induced DNA synthesis was completely abolished. 14C-Leucine incorporation was impaired in calcium-free medium but the stretch-induced increase still remained. The results suggest that mechanical force may play an important role in DNA synthesis and protein metabolism of vascular smooth muscle.

Assignment of the contribution of the tryptophan residues to the circular dichroism spectrum of human carbonic anhydrase II.

The circular dichroism (CD) spectrum of human carbonic anhydrase II (HCAII) has been investigated using various mutants of the enzyme in which tryptophans have been replaced by site-directed mutagenesis. HCAII contains seven tryptophans which are believed to significantly contribute to the CD spectrum in both the near- and far-UV regions. By substituting the tryptophans one at a time, the spectral effects of the individual tryptophans were studied. The near-UV spectrum of HCAII is very complex, with multiple Cotton effects. This complexity has been attributed to aromatic amino acids, especially tryptophans, located in asymmetric aromatic clusters in the molecule. CD spectra of the individual tryptophans were calculated as difference spectra between the CD spectrum of HCAII and those of the tryptophan mutants. These spectra showed that the tryptophans contributed to the CD spectrum in almost the entire wavelength region investigated (180-310 nm). Summation of the individual tryptophan CD spectra in the near-UV region yielded a spectrum that was qualitatively very similar to that of HCAII, showing that the tryptophans are the major determinant for this part of the CD spectrum. Since tryptophans were also demonstrated to contribute significantly in the far-UV region, tryptophans can interfere considerably with the assignment of changes in CD bands to changes in secondary structure content during folding reactions. Moreover, because of this substantial interference, predictions of the amount of various types of secondary structure from CD data from the far-UV region are made more difficult. These findings are probably of general importance for proteins that, like HCAII, contain several tryptophans.(ABSTRACT TRUNCATED AT 250 WORDS)

Folding and stability of the N-terminus of human carbonic anhydrase II.

Truncations and mutations in the N-terminus of human carbonic anhydrase II were constructed in order to establish what role this part of the protein plays in the folding and stability of the protein. When incubated in various concentrations of guanidine hydrochloride (GuHCl), HCAII unfolds in two transitions, with an intermediate state at about 1.3 M GuHCl. N-Terminal truncations of 5, 17, or 24 amino acid residues destabilize the native state by 4-5 kcal/mol, relative to the intermediate state, but these amino acid residues have virtually no effect on the stability of the intermediate state relative to the unfolded state. These truncated variants of HCAII still have a high enzymatic activity. Deletion of 28 or more amino acid residues, however, results in inactive enzyme variants. The rates at which the active site is formed are practically unaffected by the removal of the 24-amino acid segment, i.e., the active site forms independently of the N-terminus. By using the tryptophans in positions 5 and 16 as intrinsic probes, we conclude that the structure of the N-terminal region is formed very late in folding. The results strongly indicate that this process is dependent on the prior formation of an enzymatically active native-like structure of the rest of the protein.

High-resolution probing of local conformational changes in proteins by the use of multiple labeling: unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes.

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 A from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 A from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At approximately 1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 A in diameter depending on the experimental conditions and spectroscopic technique used.

Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance.

The spin-labeling method was used to investigate human carbonic anhydrase, HCA II, undergoing unfolding induced by guanidine-HCI (Gu-HCI). The spin-probe, N-(2,2,5,5-tetramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzyme. The electron paramagnetic resonance spectrum of the folded structure showed the characteristic slow motional spectra. When the concentration of the denaturing agent, Gu-HCI, was gradually increased, new spectral components with narrower lines evolved to give complex electron paramagnetic resonance spectra, apparently containing superimposed contributions from several components of different mobility. By a differentiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCI concentration. The amplitude of difference spectra versus Gu-HCI concentration showed two distinct maxima, indicating the existence of a folding intermediate state/structure. The results were found to agree with optical absorption data, which showed similar transitions at the same Gu-HCI concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, folding intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCI were obtained by least squares fitting of the simulated spectra to the experimental ones. The folding intermediate was found to have a maximum population of 39 +/- 4% at approximately 0.7 M Gu-HCI.

Antagonistic effect of amiloride on alpha-2 adrenoceptor-mediated pigment aggregation: pharmacological heterogeneity between B-HT 920 and noradrenaline.

Pigment aggregation in melanophores from the cuckoo wrasse (Labrus ossifagus L.) has previously been shown to be mediated by alpha-2 adrenoceptors. In the present investigation, the effect of amiloride on pigment aggregation induced by noradrenaline and B-HT 920 was studied. Amiloride caused a parallel shift to the right in concentration-response curves for 5-allyl-2-amino-5,6,7,8-tetrahydrothiazolo-[4,5-d]azepine-dihyd roc hloride: (B-HT 920), with no change in the maximal response, indicating simple competitive inhibition. Subsequent Schild plot analysis gave a straight line with a slope of almost unity (0.95) and a KB of 5 x 10(-6) M. In contrast to the effect of amiloride on B-HT 920-induced pigment aggregation, only a very high concentration of amiloride (1 x 10(3) M) inhibited the corresponding effect of noradrenaline. Na+/H+ exchange has been suggested as a possible mechanism for alpha-2 adrenoceptors, and the amiloride analogs 5-(N,N-dimethyl)amiloride and 5-(N,N-hexamethylene)amiloride are known to be more specific than amiloride itself in this respect. However, these analogs inhibited the effect of B-HT 920 significantly less effectively than amiloride. Furthermore, manipulating the extracellular pH between 6.8 and 7.8 did not affect the concentration response curves of B-HT 920. Neither did the prostanoid pathway inhibitors, quinacrine or indomethacin, inhibit the pigment-aggregating effect of B-HT 920. The present results suggest that amiloride competes with B-HT 920 as receptor antagonist, and that Na+/H+ exchange is insignificant for the alpha-2 adrenoceptor-stimulated pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of odorants on pigment aggregation and cAMP in fish melanophores.

Odor perception within olfactory neuroepithelium and pigment translocation within melanophores both seem to rely on a cAMP-based second messenger system. From studies on cultured frog melanophores, Lerner et al. (Proc. Natl. Acad. Sci. USA 85:261-264, 1988) suggested that some aspect of odor perception may be mediated by a nonspecific mechanism whose signal is transduced by a cAMP-based second messenger system. In the present study, odorants (beta-ionone, benzylaldehyde, cineole, cinnamaldehyde, and octanol), which previously have been shown to stimulate formation of cAMP in the olfactory neuroepithelium, were investigated for possible pigment dispersing and cAMP-increasing effects. Pretreatment of fish melanophores with the adenylate cyclase activator forskolin (1 microM) resulted in an approximately 300% increase in cAMP and an almost complete blockage of noradrenaline-induced pigment aggregation. However, none of the tested odorants were able to increase the cAMP level and only cinnaldehyde and beta-ionone were found to have any pigment dispersing activity.

Characterization of folding intermediates of human carbonic anhydrase II: probing substructure by chemical labeling of SH groups introduced by site-directed mutagenesis.

By measurement of UV absorbance, CD spectra, and enzyme activity, we have shown that human carbonic anhydrase II forms a stable and compact folding intermediate at a moderate concentration of guanidine hydrochloride. The major aim of this study was to map the intermediate structure. For that reason, site-directed mutagenesis was used to introduce cysteine residues in various parts of the central beta-structure to give in each case a single cysteine residue. Thereafter, the accessibility of the introduced SH group to specific chemical labeling was used to probe the stability and compactness of the area surrounding each cysteine residue. Our results indicate that the folding intermediate has an ordered native-like secondary structure in the central part of the beta-sheet, whereas the peripheral part of the beta-sheet seems to be less ordered. A large hydrophobic cluster situated between the central beta-sheet core and secondary structure elements on the surface appears to be intact in the intermediate and is remarkably stable even at high GuHCl concentrations (> 5 M). This unusually stable substructure might function as a "seed" during the initiation of the folding process.

Contribution of individual tryptophan residues to the fluorescence spectrum of native and denatured forms of human carbonic anhydrase II.

Measurements were made of fluorescence spectra produced by pseudo-wild-type human carbonic anhydrase II and mutants in which the tryptophan residues had been replaced by phenylalanine or cysteine residues. 2D NMR spectra of 15N-labeled proteins indicated that the mutations had essentially no long range effects on structure and that the pertubations of structure in the vicinity of the mutated Trp were small. The individual contributions of the seven tryptophan residues were deduced from measurements on native proteins and on proteins subjected to various denaturing conditions. Trp97 and Trp245 are the major fluorescence emitters in the native state, contributing 52% and 38%, respectively, to the total fluorescence intensity. Comparisons of the fluorescence yield of pseudo-wild-type human carbonic anhydrase II and mutant proteins also indicate net energy transfer from Trp16 to Trp5 and from Trp192 to Trp209. The fluorescence from Trp5 is efficiently quenched by His64. In addition, acrylamide quenching of fluorescence was used to probe the environment of tryptophans in proteins incubated in 0, 1.5, and 5 M guanidine hydrochloride. The results indicate that the part of the native protein that corresponds to beta-strands 3-7 forms a compact core in a molten globule intermediate.

Structural mapping of an aggregation nucleation site in a molten globule intermediate.

Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation.

Mapping the folding intermediate of human carbonic anhydrase II. Probing substructure by chemical reactivity and spin and fluorescence labeling of engineered cysteine residues.

Several conformation-sensitive parameters have shown that human carbonic anhydrase II exists as a stable and compact equilibrium folding intermediate of molten globule type. In this study we have continued a previously initiated mapping of the intermediate structure. Cys residues were engineered, one at a time, into various regions of the protein structure, so as to obtain chemically reactive probes and handles for spectroscopic probes. These probes were used to specifically report on conformational changes accompanying the folding process. Thus, the accessibility of the introduced Cys residues to specific chemical labeling by radioactive iodoacetate was used to monitor the stability and compactness of the substructure surrounding each Cys residue. In addition, a spin-label (nitroxide radical) and a fluorescent probe (IAEDANS) were attached to the inserted SH-groups to give complementary information. The mobility of the spin-label was used to indicate local changes in structure, and the fluorophore was used to probe local changes in polarity at various stages of unfolding. Much of the predominant beta-structure, consisting of 10 beta-strands extending throughout the entire molecule, appears to be compact and largely intact in the intermediate. Thus, beta-strands 3-7, probed at positions 68, 97, 118, 123, 206, and 245, seem to have a native-like structure in the folding intermediate. In contrast, a more flexible structure is found around positions 56, 176, and 256 in the peripheral beta-strands 1, 2, and 9, showing that the stability of the secondary structure in the intermediate state is less in the outer parts of the protein. A hydrophobic region, containing beta-strands 3-5, seems to be remarkably stable and is not ruptured until strong denaturing conditions (5 M GuHCl) are applied. The stability of this hydrophobic beta-core appears to increase toward the center. This stable region is contained in the middle of a sequentially continuous antiparallel structure that spans beta-strands 2-6, suggesting that this part might represent a site where folding is initiated.