Daizo Hamada

High helicity of peptide fragments corresponding to β-strand regions of β-lactoglobulin observed by 2D-NMR spectroscopy

Backgound: Whereas protein fragments, when they are structured, adopt conformations similar to that found in the native state, the high helical propensity of β -lactoglobulin, a predominantly β -sheet protein, suggested that the fragments of β -lactoglobulin can assume the non-native helical conformation. In order to assess this possibility, we synthesized four 17–18-residue peptides corresponding to three β -strand regions and one helical region (as a control) of β -lactoglobulin and examined their conformation. Results We observed residual helicities of up to 17% in water, by far-UV CD, for all four peptide fragments. The helices could be significantly stabilized by the addition of TFE, and the NMR analyses in a mixture of 50% water/TFE indicated that helical structures are formed in the central region whereas both termini are frayed. Thus, the very same residues that form strands in the native β -lactoglobulin showed high helical preferences. Conclusion These results stand out from the current general view that peptide fragments isolated from proteins either are unfolded or adopt native-like secondary structures. The implications of the results in the mechanism of protein folding and in designing proteins and peptides are significant.

Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering

The trifluoroethanol (TFE)‐induced conformational transition of hen lysozyme was studied with the combined use of far‐UV circular dichroism (CD) and small‐angle X‐ray scattering. At pH 2.0 and 20°C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small‐angle X‐ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain‐like conformation with some remaining globularity. The TFE‐induced transition curves obtained by CD and the small‐angle X‐ray scattering measurements agreed well, consistent with a two‐state transition mechanism. A singular value decomposition analysis of Kratky plots of the small‐angle X‐ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two‐state approximation.

Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion

Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment ‘IZUMO1 putative functional fragment (IZUMO1PFF)’. Surprisingly, IZUMO1PPF still maintains binding ability on the egg surface of Cd9-/- eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1PFF-core.

Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin

We show that a series of peptides corresponding to individual beta-strands in native beta-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the beta-strands that we considered, betaA, were found to promote fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, betaI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of betaI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.

N-Terminal Phosphorylation of HP1 Promotes Its Chromatin Binding

ABSTRACT The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1αs affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1αs N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.

Amyloid Fibril Formation and Chaperone-like Activity of Peptides from αA-Crystallin

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we found that these peptides, alphaAC(70-88), the peptide corresponding to amino acids 70-88 of alphaAC (KFVIFLDVKHFSPEDLTVK), and alphaAC(71-88), suppressed the amyloid fibril formation of amyloid beta protein (Abeta). On the other hand, while alphaAC(71-88) exhibited chaperone-like activity toward insulin, alphaAC(70-88) and alphaAC(70-88)K70D promoted rapid growth of aggregates consisting of insulin and these peptides in their solution mixtures. Interestingly, we found that alphaAC(71-88) itself can also form amyloid fibrils. It is possible that the chaperone-like activity of the alphaAC peptides is potentially related to their propensity for amyloid fibril formation. Analysis of variants of the alphaAC peptides suggested that F71 is important for amyloid formation, and interestingly, this same residue has previously been found to be essential for chaperone-like activity. Amyloid fibril formation was also observed with the shorter peptide, alphaAC(70-76)K70D, showing that the ability to form amyloid fibrils is maintained even with significant deletion of the C-terminal sequence. The formation of amyloid fibril was suppressed in alphaAC(70-88), suggesting that the K70 in the substrate binding site may play a role in suppressing the amyloid fibril formation of alphaAC, which agreed with recent proposals about the presence of an aggregation suppressor in the region flanking aggregation-prone hydrophobic sequences.

Structure and Functional Characterization of Vibrio parahaemolyticus Thermostable Direct Hemolysin

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

Effect of Lipid Type on the Binding of Lipid Vesicles to Islet Amyloid Polypeptide Amyloid Fibrils

Amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are observed near pancreatic beta-cells of type II diabetics, with their presence strongly correlating with a loss of beta-cell mass and decreased pancreatic function. Although beta-cell membranes have been implicated as the likely target of amyloidogenic IAPP toxicity, interactions between membranes and IAPP in the fibrillar state have not been well characterized. In this study, turbidity measurements were conducted to provide a detailed description of the binding reaction between IAPP fibrils and lipid vesicles made from phosphatidylcholine. The kinetic data representing the rate and extent of the fibril-vesicle binding reaction are described well by an empirical double-exponential equation. The extent of binding was found to increase with increasing amyloid fibril concentration. Modification of the vesicle composition significantly altered the observed binding reaction kinetics, with the change quantified using the parameters obtained from the double-exponential fitting analysis. When the vesicles contained a significant amount of the lipid phosphatidylglycerol, substantial sedimentation of the vesicles under gravity was detected following the initial binding reaction. To rationalize the observed kinetic binding data, we developed a mesoscopic simulation model based on a hard particle representation of the species involved. In light of the observed data and simulation predictions, the potential roles of IAPP amyloid fibrils in membrane binding are discussed.

Binding of islet amyloid polypeptide to supported lipid bilayers and amyloid aggregation at the membranes.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of β-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.

EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far‐UV and near‐UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes α‐helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side‐chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8‐anilinonaphthalene‐1‐sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea‐unfolding transitions, monitored by far‐UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N‐1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea‐unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.