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Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.

Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-phenyl-indole-3-acetic acid (5-Ph-IAA) to C. elegans and demonstrated that it confers better degradation control vs the previous system by suppressing leaky degradation and inducing sharp degradation using 1,300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analog, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.

Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.

Microtubules (MTs) are polymers composed of α- and β-tubulin heterodimers that are generally encoded by genes at multiple loci. Despite implications of distinct properties depending on the isotype, how these heterodimers contribute to the diverse MT dynamics in vivo remains unclear. Here, by using genome editing and depletion of tubulin isotypes following RNAi, we demonstrate that four tubulin isotypes (hereafter referred to as α1, α2, β1 and β2) cooperatively confer distinct MT properties in Caenorhabditis elegans early embryos. GFP insertion into each isotype locus reveals their distinct expression levels and MT incorporation rates. Substitution of isotype coding regions demonstrates that, under the same isotype concentration, MTs composed of β1 have higher switching frequency between growth and shrinkage compared with MTs composed of β2. Lower concentration of β-tubulins results in slower growth rates, and the two α-tubulins distinctively affect growth rates of MTs composed of β1. Alteration of ratio and concentration of isotypes distinctively modulates both growth rate and switching frequency, and affects the amplitude of mitotic spindle oscillation. Collectively, our findings demonstrate that MT dynamics are modulated by the combination (ratio and concentration) of tubulin isotypes with distinct properties, which contributes to create diverse MT behaviors in vivo.

Microtubules (MTs) are polymers composed of α- and β-tubulin heterodimers that are generally encoded by genes at multiple loci. Despite implications of distinct properties depending on the isotype, how these heterodimers contribute to the diverse MT dynamics

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G(2) arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G(2) arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G(2) arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/DeltaC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/DeltaC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/DeltaC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5–Sfr1 and Swi5–Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5–Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5–Sfr1 works independently of another mediator complex, Rhp55–Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.

The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.

The 244-nm excited transient UV resonance Raman spectra are observed for the refolding intermediates of horse apomyoglobin (h-apoMb) with a newly constructed mixed flow cell system, and the results are interpreted on the basis of the spectra observed for the equilibrium acid unfolding of the same protein. The dead time of mixing, which was determined with the appearance of UV Raman bands of imidazolium upon mixing of imidazole with acid, was 150 micros under the flow rate that was adopted. The pH-jump experiments of h-apoMb from pH 2.2 to 5.6 conducted with this device demonstrated the presence of three folding intermediates. On the basis of the analysis of W3 and W7 bands of Trp7 and Trp14, the first intermediate, formed before 250 micros, involved incorporation of Trp14 into the alpha-helix from a random coil. The frequency shift of the W3 band of Trp14 observed for this process was reproduced with a model peptide of the A helix when it forms the alpha-helix. In the second intermediate, formed around 1 ms after the start of refolding, the surroundings of both Trp7 and Trp14 were significantly hydrophobic, suggesting the formation of the hydrophobic core. In the third intermediate appearing around 3 ms, the hydrophobicity was relaxed to the same level as that of the pH 4 equilibrium intermediate, which was investigated in detail with the stationary state technique. The change from the third intermediate to the native state needs more time than 40 ms, while the appearance of the native spectrum after the mixing of the same solutions was confirmed separately.

A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding-dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1-2 ps.

The PAF1 complex (PAF1C) is an evolutionarily conserved protein complex involved in transcriptional regulation and chromatin remodeling. How the PAF1C is involved in animal development is still not well understood. Here, we report that, in the nematode Caenorhabditis elegans, the PAF1C is involved in epidermal morphogenesis in late embryogenesis. From an RNAi screen we identified the C. elegans ortholog of a component of the PAF1C, CTR-9, as a gene whose depletion caused various defects during embryonic epidermal morphogenesis, including epidermal cell positioning, ventral enclosure and epidermal elongation. RNAi of orthologs of other four components of the PAF1C (PAFO-1, LEO-1, CDC-73 and RTFO-1) caused similar epidermal defects. In these embryos, whereas the number and cell fate determination of epidermal cells were apparently unaffected, their position and shape were severely disorganized. PAFO-1::mCherry, mCherry::LEO-1 and GFP::RTFO-1 driven by the authentic promoters were detected in the nuclei of a wide range of cells. Nuclear localization of GFP::RTFO-1 was independent of other PAF1C components, while PAFO-1::mCherry and mCherry::LEO-1 dependent on other components except RTFO-1. Epidermis-specific expression of mCherry::LEO-1 rescued embryonic lethality of the leo-1 deletion mutant. Thus, although the PAF1C is universally expressed in C. elegans embryos, its epidermal function is crucial for the viability of this animal.

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA, Author Summary Homologous recombination promotes genetic diversity in the next generation and serves as a driving force for evolution. It also provides efficient machinery for repairing DNA damage such as double-strand breaks. Homologous recombination involves DNA exchange between homologous chromosomes, which is mediated by evolutionarily conserved proteins called recombinases. It is thought that a recombinase binds to single-stranded DNA (ssDNA) to form a nucleoprotein filament called the presynaptic filament, and that this higher order structure engages in a search for homologous DNA sequences. Once a homologous duplex is found, the presynaptic filament initiates strand exchange. However, when ssDNA regions are created, they are immediately covered by replication protein A (RPA), thereby inhibiting recombinase filament formation even under conditions in which homologous recombination is appropriate. Previous studies suggested that mediator proteins help load recombinases onto ssDNA, and further studies showed that at least two mediators function together in a single recombination pathway. How these mediators coordinate recombinase loading has been unclear. We have addressed this question by reconstituting an in vitro strand exchange reaction with purified proteins including a fission yeast recombinase, Rhp51, two mediators, Rad22 and the Swi5-Sfr1 complex, and RPA. Our results indicate that Rad22 orchestrates the loading of Rhp51 onto RPA-coated ssDNA by acting as a scaffold for nucleating the recombinase filament, whereas the other mediator, Swi5-Sfr1, stabilizes and activates the filament., A test tube reconstitution of the strand exchange reaction provides further evidence that the two mediators cooperatively promote the nucleation of active recombinase-ssDNA filaments, creating a higher order structure for recombination.

Background Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored. Results A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer. Conclusions The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Background Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

紫外共鳴ラマン分光法によるタンパク質の構造変化の解明 タンパク質の機能発現に不可欠な三次構造がどのようにして組み立てられ、機能発現にあたってどのような構造変化が起こるのかを、紫外共鳴ラマン分光法（UVRR）を用いて調べた。244 nm励起のUVRR分光法では芳香族アミノ酸残基であるトリプトファン（Trp）残基とチロシン（Tyr）残基山来のバンドが選択的に検出される。ミオグロビン（Mb）一分子あたりTrp残基が2残基、Tyr残基はマッコウクジラ由来で3、ウマ由来で2残基含まれる。これらの残基をタンパク質の構造変化のプローブとしてリガンド結合及びタンパク質の折れ畳み反応に伴う構造変化を追跡した。両残基とも幾つかのバンドが現れるが、特にTrp残基では、W3バンドとW7パンドの、Tyr残基ではY9aバンド（1170-1180 cm-1）の変化に注目した。1560-1550 cm-1に現れるW3バンドは側鎖の二面角と振動数に良い相関があり、側鎖構造の情報を与える。一方、W7バンドは、フェルミ共鳴によって1360 cm-1と1340 cm-1付近に二つに分裂しており、その強度比（I 1360/I 1340，R値）は非極性環境下だと1より大きぐ極性環境下であると0.5近くまで減少するため、Trp側鎖の環境変化を見積もる指標とした。リガンド結合に伴うミオグロビンの構造変化 マッコウクジラ由来Mbの五配位還元型と六配位CO結合型のUVRRスペクトルを比較すると、W3パンドの強度変化及びY98バンドの低波数シフトが観察され、分子量17,000の単量体タンパク質であるMbにおいてもリガンド結合に伴う構造変化があることが明らかになった。二つあるTrp残基をそれぞれフェニルアラニン（Phe）に置換した変異体，W14F，W7Fを作成した。それを用いて、変化のあったTrp残基を同定したところ、N末端に近い7番目のTrp残基の環境及び構造が変化していることが示唆された。一方、ウマ由来のMbではパクパンドの変化は同様にあったものの、Y98バンドの変化はみられなかったことから、Tyrパンドの変化は、マッコウクジラにのみあるTyr151由来の変化であると帰属した。結合ポケット側に変化を与えるかさ高いりガンドの結合したn - プチルイソシアニド結合型でもCO結台型と同じ変化を示したことから、リガンド結合に伴うタンパク質の構造変化が、ヘムの五配位型から六配位型への変換が最も大きな構造変化の要因と考えられた。これは、ヘム鉄と共有結合している軸配位子の近位ヒスチジン（His）側から構造化化が誘起され、一方で、遠位Hisの動きは局所的なものであると結論した。遠位Hisの変異体H64Y、F46V/H64Yの性質 遠位Hisとその周辺のヘムポケットを形成する残基は、二原子分子の親和性に大きく関与している。この遠位HisをTyr残基に置換したH64Y変異体は自動酸化速度が非常に早く、64番目のTyr残基がヘム鉄に配位していることが指摘されていた。実際、244 nm励起のUVRR測定によってTyr残基のプロトンが解離してチロシネート（Tyr￣）として配位していることが明らかになった。F46V/H64Y変異体はH64Yよりも高いpHで鉄から外れるが、これは遠位側残基の動き易さに起因するものと考えられた。アポミオグロビンの酸変性過程 アポミオグロビン（apoMb）は、速度論的な後期の巻き戻り中間体が、平衡条件下でも比較的安定に存在することが知られており、中間体の構造はタンパク質の構造形成を知る上で重要な情報となる。そこで酸性側（pH 5.5-1.9）の各pHで平衡状態にあるアポミオグロビン（apoMb）のUVRRスペクトルを測定し、その酸変性過程を追跡した。W3バンドは三状態変化を示し、先に変異体から求めたTrp7とTrp14の天然状態と、完全な変性状態での波数から、中間状態のスペクトレを抽出した。その結果、pH 4以上では天然状態と中間状態の二状態変化でその中点はpH 4.5、pH 4以下では中間状態と変性状態の二状態変化と考えられ、その中点はpH 3.5であった。この結果は、疎水性環境から親水性環境への変化を示すW7バンドの強度比（R値）の減少曲線とも一致した。これらの結果から、pH 4付近の平衡中間体では、すでに骨格がランダム構造になっていて、Trp7は外に露出していることが示唆された。一方、Trp14はまだ天然状態と同程度の疎水性環境下に埋もれていることが分った。W3パンドの波数から、中間状態でのTrp14の側鎖の配向け、何らかの構造制約を受けているが、天然状態とは異なることが示唆された。サブミリ秒時間領域でのアポミオグロビンの巻き戻り過程 紫外共鳴ラマン分光法に、連続フロー型高速混合装置を組み合わせて、数百マイクロ秒から数ミリ秒時間領域でのapoMbの酸変性からの巻き戻り反応を追跡した。W7バンドとW3バンドの変化から、大きく分けて3種の中間体が存在することが示唆された。1つ目は、測定時間限界の250マイクロ秒よりも早く形成するが、その状態でW3パンドは平衡論的中間体と同程度の波数シフトを示すもののまだ極性環境下にある状態で、Trp14が局所的な相互作用を受けたものと推測された。最も考えられる可能性は、Trp14が位置するAへワックスの形成であり、その後の中間体でW3バンドがシフトしなかったことから、平衡中間状態においてTrp14は非特異的な疎水性相互作用をしてタンパク質内部に埋もれているものと考えられる。2番目に現れる中間体の時定数はおよそ400マイクロ秒で一時的に疎水性環境が高まった状態である。この時Trp14同様Trp7も一時的にタンパク質内部に埋もれており、タンパク質全体が大きな疎水性コアを形成しているものと考えられる。最後に、約3ミリ秒の時定数でやや極性環境に戻るが、これはpH 4の平衡中間状態に近い。この後期の中間体から、完全な天然状態への変化は非常に遅く律速段階となっている。モデルペプチドのヘリックス形成に伴うTrp側鎖構造への影響 α ヘリックスの形成に伴い、Trp側鎖の構造を反映するW3パンドがシフトをするのかどうかを調べるために、19残基からなるAヘリックスのモデルペプチドを用いた測定を行った。このペプチドは、水溶液中ではランダムコイル構造をしているが、アルコールの添加によりヘリックス構造形成が誘起される。そこで、遊離のTrpとペプチドについて、アルコールの非存在下と存在下でのW3パンドを比較した。その結果、骨格鎖のα ヘリックス形成がTrp側鎖に影響を与え、W3パンドにシフトが起こることが実証された。これは、apoMbの巻き戻りでの早期中間体でW3パンドがシフトするのはAヘリックスが形成したためであるという考察を支持する結果である。