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The iron core of Escherichia coli ferritin was reconstituted in the presence and absence of phosphate. The core formed in the presence of phosphate contained phosphate in amounts comparable to the iron content. The size distribution of the core was analyzed by analytical ultracentrifugation. A continuous size distribution was observed in the presence of phosphate, whereas a multimodal distribution was found in the absence of phosphate. In the presence of phosphate, the core size observed by electron microscopy was consistent with the inner diameter of ferritin. In contrast to this, clusters of several smaller particles were observed in the absence of phosphate. The small-angle X-ray scattering was measured under contrast matching conditions to obtain information on the iron core shape. A fringe was observed in the scattering profile in the presence of phosphate, but it was not observed in the absence of phosphate. Combining all results, we conclude that a hollow spherical core was formed in the presence of phosphate, while several small particles were formed within the inner cavity in the absence of phosphate.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family, and it mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia is composed of the passenger domain, which is a virulence factor, and the translocator domain, which anchors the passenger domain into the outer membrane. The Hia transmembrane domain forms a transmembrane β-barrel of 12 β-strands, four of which are provided from each subunit. The β-barrel has a pore that is traversed by three α-helices, one of which is provided from each subunit. This domain has a unique arginine arrangement inside the β-barrel. The side chains of the arginine residues protrude from the β-strands of three subunits toward the center of the barrel and are close to each other. Mutation of this arginine residue revealed the importance of the electrostatic repulsion between the three arginines. Electrostatic repulsion is considered to prevent misfolding and/or misassembly. The arginine clusters at the interface were found in several proteins and might generally play an important role in the assembly of the oligomer.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (SAXS). EcFtnA forms a cage-like structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains native-like secondary and tertiary structures and can reassemble into a 24-mer when the pH is increased. The time-dependent changes in the SAXS profiles of ferritin during its assembly were roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered intermediates. The rate of assembly increased with increasing ionic strength and decreased with increasing pH (from pH 6 to pH 8). These tendencies might originate from repulsion between assembly units (dimers) with the same net charge sign. To test this hypothesis, ferritin mutants with different net charges (net-charge mutants) were prepared. In buffers with low ionic strength, the rate of assembly increased with decreasing net charge. Thus, repulsion between the assembly unit net charges was an important factor influencing the assembly rate. Although the differences in the assembly rate among net-charge mutants were not significant in buffers with an ionic strength higher than 0.1, the assembly rates increased with increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic-strength dependence of the assembly rate and are, on average, repulsive.

Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement disorder caused by mutations in the ferritin gene. A ferritin light-chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism, analytical ultracentrifugation, and small-angle X-ray scattering studies have shown that both the subunit structure and the assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). The iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defects observed for A96T were a lower refolding efficiency and a stronger propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.

To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability. As a result, the amount of purified HiaTD reached a plateau at 2 h postinduction. Although expression without the signal sequence provides a large amount of proteins as inclusion bodies in some membrane proteins, HiaTD expressed without a signal sequence was not observed as inclusion bodies and seemed to be assembled into the outer membrane during or after cell lysis.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family and mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia contains a passenger and a transmembrane domain. The transmembrane domain forms a 12-stranded β-barrel in which four strands are provided by each subunit. The β-barrel has a pore that is traversed by three α-helices. This domain has a unique arginine cluster, in which the side chains of the three arginine residues located at position 1077 (Arg1077) protrude into the pore of the β-barrel. This arrangement seems to be unfavorable for assembly, because of repulsion between the positive charges. In this study, we investigated the in vitro assembly of the Hia transmembrane minimum domain (mHiaTD) and found that the dissociated mHiaTD reassembled in detergent solution. To investigate the role of Arg1077 in trimer assembly, we generated mutant proteins in which Arg1077 was replaced with methionine or lysine. The reassembly kinetics of the mutants was compared with that of the wild-type protein. The methionine mutant showed misassembly, whereas the lysine mutant showed reversible assembly, similar to that observed for the wild-type protein. These results show that electrostatic repulsion between the positive charges of Arg1077 is important for preventing the formation of misassembled oligomers by the mHiaTD in vitro.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine β-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 651–658, 2014.

β-Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat β-sheets, suggesting that side chains influence β-structures. We therefore investigated the relationship between amino acid composition and twists or bends of β-strands. We calculated and statistically analyzed the twist and bend angles of short frames of β-strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of β-strands, suggesting that the side chains of these residues likely do not affect β-strand structure. In contrast, the majority of serine, threonine, and asparagine side-chains in β-strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress β-strand twisting.

In 1996, Ballew et al.1 reported that, according to laser T-jump (up) measurements from the cold denatured state, apomyoglobin is folded in three phases; the single α-helix (helix A) formation (1 μs), the collapse of α-helices (A, G and H; 5–17 μs) and folding to its final conformation (0.5 s), and emphasized that the initial collapse of protein folding took place in the μs or even faster timescale. In 1999, Clarke et al.2 reported a rate of α-helix formation as slow as 60 ms in the case of oligopeptide AK16 (composed of 16 amino acid residues) by means of the GuHCl concentration-jump, using a stopped-flow method. They attributed the difference to the laser T-jump study in which Ballew et al. employed only shifted equilibrium. The initial state already included small fractions of α-helices, and therefore, the rate that Ballew et al. observed was not the nucleation rate of α-helix but rather the propagation rate of the α-helix. They also measured the rate of α-helix formation of polyglutamic acid and poly-L-lysine, and reported that the rates of such long polypeptides are observable but faster than those of short peptides. As to the reason, they speculated that the long peptides have some nuclei of α-helix even in the 4 M GuHCl. We recently found that the initial step of α-helix formation in bovine β-lactoglobulin finished within the dead time of the stopped-flow apparatus (6 ms) at 4°C3. This result was reproduced with other proteins such as SH3 domain4, FHA domain5 and equine β-lactoglobulin6. Our results cannot be understood if the initial α-helix formation rate is as slow as Clarke et al. reported. Therefore, we started measuring the nucleation rate of oligopeptide, C17 (DLTDDIMCVKKILDKVG), α-helical part of 84th to 100th amino acids of bovine α-lactalbumin7. We found only initial bursts of the increase of α-helices at temperatures higher than −30°C in the presence of 70% methanol. A similar experiment was conducted with AK16 peptide, as Clarke had used in his experiment. In addition, we measured the folding rate of polyglutamic acid at 4°C. The result only showed the initial burst. These results strongly indicate that the α-helix formation rates in the case of C17 and AK16 peptides and polyglutamic acid are faster than the dead time of the stopped-flow apparatus (6 ms). The difference between our and Clarkes’ data is discussed.

Protein folding is a thermodynamic process driven by energy gaps between the native and unfolded states. Although a wealth of information is available on the structure of folded species, there is a paucity of data on unfolded species. Here, we analyzed the structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger (SBD) formed in the presence of guanidinium hydrochloride (GuHCl). Although far-UV CD and intrinsic tryptophan fluorescence spectra as well as small angle X-ray scattering suggested that SBD assumes highly unfolded structures in the presence of GuHCl, near-UV circular dichroism of wild-type SBD suggested the presence of residual structures in the unfolded state. Analyses of the unfolded states of tryptophan mutants (W543L, W563A, W590A and W615L) using Similarity Parameter, a modified version of root mean square deviation as a measure of similarity between two spectra, suggested that W543 and W563 have preferences to form native-like residual structures in the GuHCl-unfolded state. In contrast, W615 was entirely unstructured, while W590 tended to form non-native ordered structures in the unfolded state. These data and the amino acid sequence of SBD suggest that local structural propensities in the unfolded state can be determined by the probability of the presence of hydrophobic or charged residues nearby tryptophan residues.

Equine β-lactoglobulin is a 162-residue β-sheet protein. A partially folded form of equine β-lactoglobulin contains a β-hairpin and an α-helix. The β-hairpin converts into non-native α-helices at temperatures0°C. CHIBL, a truncated equine β-lactoglobulin (residues 88-142), contains the low-temperature α-helical structure even at room temperature, indicating that the interactions responsible for the stability of the β-hairpin reside in non-CHIBL residues. For the study reported herein, we characterized two truncated mutants and their leucine103 → proline103 variants to identify residues that stabilize the β-hairpin. The dependence of their circular dichroism spectra on chloride ion concentration and temperature revealed that the ability to transition from the non-native α-helices to the β-hairpin depends on the polypeptide chain length and improves as the chain length increases despite the apparent absence of any ordered structure in the extended sequences.

Escherichia coli non-heme-binding ferritin A (EcFtnA) is a spherical cagelike protein that is composed of 24 identical subunits. EcFtnA dissociates into 2-mers under acidic conditions and can reassemble into the native structure when the pH is increased. To understand how electrostatic interactions influence the assembly reaction, the dependence of the process on ionic strength and pH was investigated. The assembly reaction was initiated by stopped-flow mixing of the acid-dissociated EcFtnA solution and high-pH buffer solutions and monitored by time-resolved small-angle X-ray scattering. The rate of assembly increased with increasing ionic strength and decreased with increasing pH from 6 to 8. These dependences were thought to originate from repulsion between assembly units (2-mer in the case of EcFtnA) with the same net charge sign; therefore, to test this assumption, mutants with different net charges (net-charge mutants) were prepared. In buffers with a low ionic strength, the rate of assembly increased with a decreasing net charge. Thus, repulsion between the assembly unit net charges was demonstrated to be an important factor determining the rate of assembly. However, the difference in the assembly rate among net-charge mutants was not significant in buffers with an ionic strength of >0.1. Notably, under such high-ionic strength conditions, the assembly rate increased with an increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic strength dependence of the rate of assembly and are repulsive on average.

β-Lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.

β-lactoglobulin (LG) contains nine β-strands (strands A–I) and one α-helix. Strands A–H form a β-barrel. At neutral pH, equine LG (ELG) is monomeric, whereas bovine LG (BLG) is dimeric, and the I-strands of its two subunits form an intermolecular β-sheet. We previously constructed a chimeric ELG in which the sequence of the I-strand was replaced with that of BLG. This chimera did not dimerize. For this study, we constructed the new chimera we call Gyuba (which means cow and horse in Japanese). The amino acid sequence of Gyuba includes the sequences of the BLG secondary structures and those of the ELG loops. The crystal structure of Gyuba is very similar to that of BLG and indicates that Gyuba dimerizes via the intermolecular β-sheet formed by the two I-strands. Thus, the entire arrangement of the secondary structural elements is important for LG dimer formation.

Tear lipocalin and β-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of β-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence. Proteins 2009. © 2008 Wiley-Liss, Inc.

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.

The secondary structure formed in disulfide reduced alpha-lactalbumin is investigated by CD spectrum and is compared with that of the folding intermediate of the disulfide intact protein. The peptide backbone structure of the reduced protein depends strongly on salt concentration in contrast to that of the intermediate. It is close to a random coil in the absence of salt, but it is almost the same as that of the intermediate at a high concentration of salt. The secondary structures of both the proteins undergo broad unfolding transitions when temperature is raised or when urea is added. The secondary structure of the reduced protein is less stable against both heat and urea. These results show that the disulfide bonds are not a determinant of the secondary structure formed at an early stage of folding, and they stabilize the secondary structure of the folding intermediate.

Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, which is similar to the helix-rich state of ELG at pH 4.0. A kinetic study was done on the folding rate of ELG and compared with bovine beta-lactoglobulin (BLG). Transient intermediates, which were observed as the burst phase of the refolding reaction, were also rich in alpha-helix. The activation enthalpy of ELG was calculated to be c.a. 80 kJ/mol, whereas that of BLG was c.a. 70 kJ/mol in the presence of 45% EGOH. The ellipticities of the transient intermediate of ELG show temperature dependence in the presence of 45% EGOH, whereas that of BLG did not show significant dependence. This study therefore extends the existence of helix-rich equilibrium and transient intermediates of predominantly beta-sheet proteins to alkaline buffer conditions.

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Background Many β-strands are not flat but bend and/or twist. However, although almost all β-strands have a twist, not all have a bend, suggesting that the underlying force(s) driving β-strand bending is distinct from that for the twist. We, therefore, investigated the physical origin(s) of β-strand bends. Methods We calculated rotation, twist and bend angles for a four-residue short frame. Fixed-length fragments consisting of six residues found in three consecutive short frames were used to evaluate the twist and bend angles of full-length β-strands. Results We calculated and statistically analyzed the twist and bend angles of β-strands found in globular proteins with known three-dimensional structures. The results show that full-length β-strand bend angles are related to the nearby aromatic residue content, whereas local bend angles are related to the nearby aliphatic residue content. Furthermore, it appears that β-strands bend to maximize their hydrophobic contacts with an abutting hydrophobic surface or to form a hydrophobic side-chain cluster when an abutting hydrophobic surface is absent. Conclusions We conclude that the dominant driving force for full-length β-strand bends is the hydrophobic interaction involving aromatic residues, whereas that for local β-strand bends is the hydrophobic interaction involving aliphatic residues. Electronic supplementary material The online version of this article (doi:10.1186/s12900-015-0048-y) contains supplementary material, which is available to authorized users.

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

At neutral pH, equine β-lactoglobulin (ELG) is monomeric, whereas bovine β-lactoglobulin (BLG) exists as a dimer. To understand the difference in the oligomerization properties between ELG and BLG, three mutants of ELG (LP, I, and LPI) were constructed by substituting amino acids responsible for important interactions at the dimer interface of BLG into ELG. The mutant LP has an AB loop mutation (S34A/E35Q), the mutant I has an I strand mutation (G145M/R146H/V147I/Q148R/I149L/V150S/P151F/D152N/L153P) and the mutant LPI includes both the LP and I mutations. The far- and near-UV CD spectra of the three mutants are similar to that of the wild-type ELG, indicating that the secondary and the tertiary structures of ELG are not significantly affected by the mutations. Ultracentrifuge analysis shows that all three mutants are monomeric at neutral pH, suggesting that the protein sequences in the AB loop and I strand of BLG alone cannot support dimerization of ELG. Thus, structural differences must exist between ELG and BLG that prevent the ELG mutants from forming the same interactions as BLG at the dimer interface. Proteins 2002;49:297–301. © 2002 Wiley-Liss, Inc.

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

In ideal proteins, only native interactions are stabilized step-by-step in a smooth funnel-like energy landscape. In real proteins, however, the transient formation of non-native structures is frequently observed. In this review, the transient formation of non-native structures is described using the non-native helix formation during the folding of b-lactoglobulin as a prominent example. Although b-lactoglobulin is a predominantly b-sheet protein, it has been shown to form non-native helices during the early stage of folding. The location of non-native helices, their stabilization mechanism, and their role in the folding reaction are discussed.

β-Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat β-sheets, suggesting that side chains influence β-structures. We therefore investigated the relationship between amino acid composition and twists or bends of β-strands. We calculated and statistically analyzed the twist and bend angles of short frames of β-strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of β-strands, suggesting that the side chains of these residues likely do not affect β-strand structure. In contrast, the majority of serine, threonine, and asparagine side-chains in β-strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress β-strand twisting.

The molten globule structure of equine beta-lactoglobulin has been inferred from the hydrogen exchange protection of the backbone amide protons. In order to make it possible to measure the hydrogen exchange kinetics of the individual backbone amide protons, the uniformly (15)N-labeled recombinant protein was expressed in Escherichia coli and the NMR peak assignment was obtained for most of the backbone protons. The chemical shift and NOE results obtained under the condition where the protein assumes the native structure are fully consistent with the known secondary structure of bovine beta-lactoglobulin, indicating that the equine protein has a similar native conformation to that of the bovine protein. The hydrogen exchange rate of the individual backbone amide protons was measured under the conditions where the protein assumes the native and molten globule states. In the native state, strong protection was observed for the residues located in the eight (A to H) strands, which form a barrel structure, and residues of a major helix. In the molten globule state at acidic pH conditions, significant protection from the exchange has been observed for residues located in the A, F, G and H strands in the native structure. The pattern of protection is consistent with a native-like beta-sheet formation by these strands. The residues located in a major helix of the native structure are also protected, suggesting that this helix is formed in the molten globule and is packed against the sheet as in the native structure. These results indicate that a native-like subdomain is formed in the molten globule state of equine beta-lactoglobulin.

Properties of three equine milk proteins, lysozyme, α-lactalbumin, and β-lactoglobulin, were investigated and compared with the corresponding ones of bovine milk. The subsite structure similar to that in hen egg lysozyme was assumed in the functional region of lysozyme from interaction with inhibitors. α-lactalbumin has structures similar to those of bovine, but less stability of its intact and 3SS forms are observed than the corresponding bovine irrespective of [Ca 2+ ] because of the entropy factors. Although the folding of β-lactoglobulin at acid pH is very similar to that of the bovine protein the conformation of the I strand is different from that of bovine I strand at neutral pH according to NMR data.

For the construction of an overexpression system of the intracellular 1,2-alpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1, 2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-alpha-d-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manalpha1-3[Manalpha1-2Manalpha1-3(Manalpha1-6) Manalpha1-6]Manbeta1- 4GlcNAcbeta1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1.cm-1. Contents of the secondary structure (alpha-helix, beta-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 degrees C by differential scanning calorimetry. The calorimetric enthalpy, DeltaHcal, of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.

The binding constants between disulfide-intact or various disulfide-reduced bovine alpha-lactalbumins and an Escherichia coli chaperonin, GroEL, were determined by using the equilibrium dialysis method. The disulfide-intact and one-disulfide (Cys6-Cys120)-reduced alpha-lactalbumins were shown not to bind with GroEL both in the presence and absence of Ca2+. The two-disulfide (Cys6-Cys120 and Cys28-Cys111)-reduced alpha-lactalbumin, which has the native-like tertiary structure in its beta-domain region and an unfolded alpha-domain in the presence of Ca2+, showed considerable binding with GroEL. The binding free energy of the two-disulfide-reduced alpha-lactalbumin in the presence of Ca2+ is close to that of the molten globule state of disulfide-intact alpha-lactalbumin. This result suggests that GroEL binds to the unfolded alpha-domain of alpha-lactalbumin regardless of the conformation of the beta-domain. The fully disulfide-reduced and two-disulfide-reduced alpha-lactalbumins were found to bind more strongly with GroEL in the absence of Ca2+ than the two-disulfide-reduced alpha-lactalbumin in the presence of Ca2+, thus indicating that the unfolding of the beta-domain of alpha-lactalbumin leads to stronger interaction with GroEL.

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.

The acid-unfolded state of equine beta-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine beta-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine beta-lactoglobulin is a molten globule state. The alpha helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative alpha helices in the molten globule state. This result suggests the generality of the intermediate with nonnative alpha helices during the folding of proteins having the beta-clam fold.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

We investigated the conformations of peptides that encompass the B helix or C helix region formed in the molten globule of bovine alpha-lactalbumin to get information on the molecular mechanism that stabilizes the molten globule. The CD spectra show that the isolated B and C helices are intrinsically unstable. The chemical shifts, NOE connectivities, and CD spectrum indicate that no helical structure is induced in the C helix region (86-99) by extending the peptide sequence to include the hydrophobic cluster region (101-107), although the hydrophobic cluster region can be regarded as a possible initiation site for folding of the protein. We also clarified that the isolated B helix (23-34) peptide does not directly interact with the C helix or hydrophobic cluster region. These results suggest that the B and C helices in the molten globule are stabilized by their interaction with other parts of the protein.

Examination was made to determine the structure of a major neutral glycolipid (GL-la) from Sulfolobus acidocaldarius N-8. NMR indicated GL-la to have a structure in which a glucose is linked via a glycosidic bond at the β-position to the ring of calditol, 2-hydroxymethy1-1- (2, 3-dihydroxypropoxy) -2, 3, 4, 5-cyclopentanetetraol, in calditoglycerocaldarchaeol (caldityl-glyceryl-dialkyl-tetraether core lipid).

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.

β-lactoglobulin (LG) contains nine β-strands (strands A–I) and one α-helix. Strands A–H form a β-barrel. At neutral pH, equine LG (ELG) is monomeric, whereas bovine LG (BLG) is dimeric, and the I-strands of its two subunits form an intermolecular β-sheet. We previously constructed a chimeric ELG in which the sequence of the I-strand was replaced with that of BLG. This chimera did not dimerize. For this study, we constructed the new chimera we call Gyuba (which means cow and horse in Japanese). The amino acid sequence of Gyuba includes the sequences of the BLG secondary structures and those of the ELG loops. The crystal structure of Gyuba is very similar to that of BLG and indicates that Gyuba dimerizes via the intermolecular β-sheet formed by the two I-strands. Thus, the entire arrangement of the secondary structural elements is important for LG dimer formation.

The urea-induced unfolding of the molten globule state of bovine alpha-lactalbumin was investigated by 1H nuclear magnetic resonance. In the molten globule state, most of the aromatic resonances deviate from their random coil values, indicating that aromatic side chains form some ordered structures in the molten globule state. When the urea concentration increases, the resonances are shifted, and the deviations from the random coil values are diminished. Because the chemical shifts of several random coil peptides are found to be independent of urea concentration, the urea-induced shifts of the resonances in the molten globule state reflect the unfolding transition of some ordered structures. The unfolding transitions measured by individual aromatic resonances do not coincide with each other. The unfolding transition curves obtained from some aromatic resonances are also different from those of the secondary structures measured by circular dichroism spectra. These results clearly show that the unfolding of the molten globule state of alpha-lactalbumin is not a cooperative two-state process.

We already reported that 281 bp DNA was degraded to 5'-dNMP by treatment at 70 degrees C and pH 7.5 for 1 h in the presence of 10 mM Mn ions, and the detailed results are published on Biosci. Biotechnol. Biochem., Vol. 71, 2670-2679 (2007). The degradation was accelerated by 100 mM NaCl. More than 80 bp DNA prepared by PCR using human ZNF 219 cDNA as the template were degraded into 5'- dNMP. Fifty bp DNA prepared by PCR us- ing the synthetic F and R primers of 22 mer was degraded into unknown material besides dNMP. Single-strand 281 b DNA prepared by Strandase (Novagen) was suggested not to be degraded into dNMP but to be degraded into the unknown material. Only double-strand DNA is presumed to be degraded into dNMP, therefore the double-strand structure is considered to be necessary for the degradation into dNMP. Furthermore, the unknown material was found in the ppt. fraction after centrifugation of the reaction mixture in the case of 34mer only G oligomer, while 5'-dGMP were found not to be degraded into any material. The m/z of the unknown material prepared from 34mer only G oligomer was determined to be 266 by LC-TOFMS. The elucidation of the conversion mechanism is under investigation.

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.

The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefore, the backbone topology in the folding core is significantly different from that in the native structure. This indicates that the burst-phase folding intermediate of beta-lactoglobulin is a trapped species because of misfolded backbone topology.

Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native alpha-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.