Induction of stalk and spore cell differentiation by cyclic AMP in slugs of Dictyostelium discoideum.

Multicellular masses of the cellular slime mold Dictyostelium discoideum, under conditions which ordinarily suppress cell differentiation, develop clusters of stalk cells and spore cells when implanted with Sephadex particles that had been soaked in 5 X 10(-3)M cyclic adenosine monophosphate (AMP). A possible relation exists between oxygen gradients, cyclic AMP gradients, and the pattern of morphogenesis and cell differentiation during fruiting.

Conversion of bacteriorhodopsin into a chloride ion pump.

In the light-driven proton pump bacteriorhodopsin, proton transfer from the retinal Schiff base to aspartate-85 is the crucial reaction of the transport cycle. In halorhodopsin, a light-driven chloride ion pump, the equivalent of residue 85 is threonine. When aspartate-85 was replaced with threonine, the mutated bacteriorhodopsin became a chloride ion pump when expressed in Halobacterium salinarium and, like halorhodopsin, actively transported chloride ions in the direction opposite from the proton pump. Chloride was bound to it, as revealed by large shifts of the absorption maximum of the chromophore, and its photointermediates included a red-shifted state in the millisecond time domain, with its amplitude and decay rate dependent on chloride concentration. Bacteriorhodopsin and halorhodopsin thus share a common transport mechanism, and the interaction of residue 85 with the retinal Schiff base determines the ionic specificity.

Repeated family of genes controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of any one of five unlinked genes: MAL1, MAL2, MAL3, MAL4, or MAL6. Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. The MAL genes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from a MAL6 strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two cryptic MAL genes in CB11, MAL1g and MAL3g (linked to MAL1 and to MAL3, respectively), in addition to the MAL6 locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected three HindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of the MAL loci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked to MAL6. Since the MAL6 locus has previously been shown to contain a regulatory gene, the MAL6 locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to the MAL6-derived probe shows that either MAL2 and MAL4 are not related to MAL6, or the DNA corresponding to these genes is absent from the MAL6 strain CB11.

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.

Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

Constitutive expression of the maltose fermentative enzymes in Saccharomyces carlsbergensis is dependent upon the mutational activation of a nonessential homolog of MAL63.

Maltose fermentation in Saccharomyces carlsbergensis is dependent upon the MAL6 locus. This complex locus is composed of the MAL61 and MAL62 genes, which encode maltose permease and maltase, respectively, and a third gene, MAL63, which codes for a trans-acting positive regulatory product. In wild-type strains, expression of the MAL61 and MAL62 mRNAs and proteins is induced by maltose and induction is dependent upon the MAL63 gene. Mutants constitutively expressing the MAL61 and MAL62 gene products have been isolated in mal63 backgrounds, and the mutations which have been analyzed map to a fourth MAL6-linked gene, MAL64. Cloning and characterization of this new gene are described in this report. The results revealed that the MAL64-C alleles present in constitutive strains encode a trans-acting positive function required for constitutive expression of the MAL61 and MAL62 gene products. In inducible strains, the MAL64 gene is dispensable, as deletion of the gene had no effect on maltose fermentation or maltose-regulated induction. MAL64 encoded transcripts of 2.0 and 1.4 kilobase pairs. While both MAL64 mRNAs were constitutively expressed in constitutive strains, they were maltose inducible in wild-type strains and induction was dependent upon the MAL63 gene. The MAL63 and MAL64 genes are at least partially structurally homologous, suggesting that they control MAL61 and MAL62 transcript accumulation by similar mechanisms.

Evidence of local conformational fluctuations and changes in bacteriorhodopsin, dependent on lipids, detergents and trimeric structure, as studied by 13C NMR.

We examined how the local conformation and dynamics of [3-13C]Ala-labeled bacteriorhodopsin (bR) are altered as viewed from 13C NMR spectra when the natural membrane lipids are partly or completely replaced with detergents. It turned out that the major conformational features of bR, the alphaII-helices, are generally unchanged in the delipidated or solubilized preparations. Upon partial delipidation or detergent solubilization, however, a significant conformational change occurs, ascribed to local conversion of alphaII-->alphaI-helix (one Ala residue involved), evident from the upfield displacement of the transmembrane helical peak from 16.4 ppm to 14.5 ppm, conformational change (one or two Ala residues) within alphaII-helices from 16.4 to 16.0 ppm, and acquired flexibility in the loop region (especially at the F-G loop) as manifested from suppressed peak-intensities in cross-polarization magic angle spinning (CP-MAS) NMR spectra. On the other hand, formation of monomers as solubilized by Triton X-100, Triton N-101 and n-dodecylmaltoside is characterized by the presence of a peak at 15.5 ppm and a shifted absorption maximum (550 nm). The size of micelles under the first two conditions was small enough to yield 13C NMR signals observable by a solution NMR spectrometer, although 13C CP-MAS NMR signals were also visible from a fraction of large-sized micelles. We found that the 16.9 ppm peak (three Ala residues involved), visible by CP-MAS NMR, was displaced upfield when Schiff base was removed by solubilization with sodium dodecyl sulfate, consistent with our previous finding of bleaching to yield bacterioopsin.

The proton transfers in the cytoplasmic domain of bacteriorhodopsin are facilitated by a cluster of interacting residues.

The stepwise internal proton transfer reactions across the membrane, and the release and uptake at the surface, are the elementary steps that together constitute the transport mechanism in a proton pump. Although the proton donor and acceptor residues can be usually identified, the directionality and the energetics of the proton transfer must be determined to a large extent also by interactions of these with neighboring groups. We have examined the roles of residues D96, T46 and R227 in proton transfers during the photocycle of bacteriorhodopsin near its cytoplasmic surface, and in general the relationship between the reprotonation of the Schiff base and the subsequent proton uptake from the cytoplasmic side. The phenotypes of single and double mutants suggest close functional interaction among D96, T46, R227, and probably internal bound water. Measurements of the free energies of activation indicate that mechanistic interpretation of the rates changed by residue replacements is hindered by a general tendency toward lowered activation enthalpies in the mutated proteins. There is less ambiguity in the free energy levels of the photointermediates. It appears from these that the inhibitory and stimulatory influences of T46 and R227, respectively, on D96 as a proton donor compensate one another and ensure the effective reprotonation of the Schiff base. T46 and D96 mediate, in turn, proton uptake at the cytoplasmic surface. Although ultimately this will reprotonate D96, the observation of proton uptake from the bulk in R82Q without reprotonation of the aspartate residue suggests that the direct proton acceptor is not D96. The results thus indicate that the passage of the proton from the surface to the Schiff base is facilitated by multiple residue and water interactions in the cytoplasmic domain.

Transient channel-opening in bacteriorhodopsin: an EPR study.

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.

Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin.

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin.

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.

Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.

Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.

Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin.

The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.

Removal of Mig1p binding site converts a MAL63 constitutive mutant derived by interchromosomal gene conversion to glucose insensitivity.

Maltose fermenting strains of Saccharomyces cerevisiae have one or more complex loci called MAL. Each locus comprises at least three genes: MALx1 encodes maltose permease, MALx2 encodes maltase, and MALx3 encodes an activator of MALx1 and MALx2 (x denotes one of five MAL loci, with x = 1, 2, 3, 4, or 6). The MAL43c allele is constitutive and relatively insensitive to glucose repression. To understand better this unique phenotype of MAL43c, we have isolated several MAL63c constitutive mutants from a MAL6 strain. All constitutive mutants remain glucose repressible, and all have multiple amino acid substitutions in the C-terminal region, now making this region of Mal63cp similar to that of Mal43cp. These changes have been generated by gene conversion, which transfers DNA from the telomeres of chromosome II and chromosome III or XVI to chromosome VIII (MAL6). The removal of a Mig1p binding site from the MAL63c promoter leads to a loss of glucose repression, imitating the phenotype of MAL43c. Conversely, addition of a Mig1p binding site to the promoter of MAL43c converts it to glucose sensitivity. Mig1p modulation of Mal63p and Mal43p expression therefore plays a substantial role in glucose repression of the MAL genes.

An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium.

The mechanism by which bacteriorhodopsin (BR) transports protons across the cell membrane of Halobacterium halobium is actively studied in many laboratories. Currently available systems for the synthesis of mutant proteins obtained by site-directed mutagenesis of the gene encoding BR (bop) require reconstitution of the denatured polypeptide after its synthesis Escherichia coli or yeast; this approach is technically difficult and labor intensive, and raises questions about possible differences between in vivo and in vitro folding. Using a newly described transformation system and a halobacterial plasmid vector, we show that it is possible to reintroduce the bop gene into BR- strains of H. halobium. The bop-carrying plasmid expresses native BR in amounts similar to those obtained in several wild type strains. This system allows facile site-directed mutagenesis in halophilic archaebacteria.

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum.

The structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-blue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is maintained. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra.

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR.

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix.

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.

Intramembrane signaling mediated by hydrogen-bonding of water and carboxyl groups in bacteriorhodopsin and rhodopsin.

The light-induced mechanism for proton pumping of bacteriorhodopsin was studied by Fourier transform infrared spectroscopy of the discrete sequential intermediate states, L, M, and N. Attention is focused on L in the early microsecond time range, as a transition state in which the Schiff base forms strong H-bonding with a water molecule coordinated with Asp85. This structure leads to transfer of the Schiff base proton to Asp85 in the L-to-M process, which then triggers proton release from Glu204 to the extracellular surface. H-bonding of Arg82 and water molecules are involved in this process. Chloride can replace Asp85 in the D85T mutant, and this anion will be then transported instead of a proton. In L, structural perturbations are induced also around Asp96, through a string of H-bonding mediated by internal water molecules and peptide carbonyls in helices B and C, and Trp182 in helix F. These may cause the structural changes that occur later in the M-to-N process. Similar interactions, through internal water molecules and the peptide bonds in helices B and C, take place in bovine rhodopsin. They transduce changes across the membrane from the Schiff base to the cytoplasmic surface, where the activation of the transducin occurs.

Stability of the C-terminal alpha-helical domain of bacteriorhodopsin that protrudes from the membrane surface, as studied by high-resolution solid-state 13C NMR.

We have recorded 13C NMR spectra of [1-(13)C]Ala- and [3-(13)C]Ala-bacteriorhodopsin (bR), [1-(13)C]Ala- and [3-(13)C]Ala-papain-cleaved bR, and [3-(13)C]Ala-labeled R227Q bR mutant by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. The pH and temperature were varied, and Arg 227 was replaced with Gln (R227Q), in order to clarify their effects on the stability of the alpha-helical domain of the C-terminus that protrudes from the membrane surface. The comparative 13C CP- and DD-MAS NMR study of [3-(13)C]Ala-bR, rather than [1-(13)C]Ala-bR, turned out to be the best means to distinguish the 13C NMR signals of the C-terminus from those of the rest of the transmembrane helices or loops. The inner segment of the C-terminus, from Ala 228 to Ala 235, forms an alpha-helical domain (resonated at 15.9 ppm) either at neutral pH and/or at 10 to -10 degrees C. The alpha-helical peak was not seen, however, after either cleavage of the C-terminus with papain or lowering the pH to 4.25. This alpha-helical structure, and a part of the random coil which was produced from the helix at pH 4.25, were further converted to a low-temperature-type alpha-helix, as indicated by an upfield displacement of the 13C NMR signal, when the temperature was lowered to 10- -10 degrees C. Surprisingly, the corresponding helical structure in R227Q is more stable than in the wild type at the acidic pH. This alpha-helical peak was classified as an alphaII-helix from the 13C chemical shifts of Cbeta carbon, although it was ascribed to an alphaI-helix on the basis of the carbonyl shifts. This is in contrast to Ala 53 which adopts the alphaII-helix as judged from the 13C chemical shifts of Cbeta and the carbonyl carbons. Therefore, this discrepancy might be caused by differential sensitivity of the two types of carbon signals to conformation and to modes of hydrogen bonding when motional fluctuation is involved. It is likely that the alphaII-helix form present at the C-terminus is not always the type originally proposed but should be considered as a form undergoing large-amplitude conformational fluctuation around alpha-helix.