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The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5&ndash, 10 min) formation of large (&gt, 2 &mu, m) aggregates. sMyBP-C oligomers formed both at the initial 5&ndash, 10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7&ndash, 10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-&beta, quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (&lt, 26%), alternating ordered/disordered regions in the protein molecule, and S&ndash, S bonds providing for general stability.

Sirtuin4 (Sirt4) is one of the mammalian homologues of Silent information regulator 2 (Sir2), which promotes the longevity of yeast, C. elegans, fruit flies and mice. Sirt4 is localized in the mitochondria, where it contributes to preventing the development of cancers and ischemic heart disease through regulating energy metabolism. The ADP-ribosylation of glutamate dehydrogenase (GDH), which is catalyzed by Sirt4, downregulates the TCA cycle. However, this reaction mechanism is obscure, because the structure of Sirt4 is unknown. We here constructed structural models of Sirt4 by homology modeling and threading, and docked nicotinamide adenine dinucleotide+ (NAD+) to Sirt4. In addition, a partial GDH structure was docked to the Sirt4-NAD+ complex model. In the ternary complex model of Sirt4-NAD+-GDH, the acetylated lysine 171 of GDH is located close to NAD+. This suggests a possible mechanism underlying the ADP-ribosylation at cysteine 172, which may occur through a transient intermediate with ADP-ribosylation at the acetylated lysine 171. These results may be useful in designing drugs for the treatment of cancers and ischemic heart disease.

A connected space is called a C0-space if its rational cup product is trivial. A characterizing property of C0-spaces is obtained. This property is used to calculate the algebraic K0-group K0(C𝔽(X)) of the ring of continuous functions for infinite-dimensional complexes X.

Let E ( X ) be the set of homotopy classes of self-homotopy equivalences of a space X. The set E ( X ) is a group by composition of homotopy classes. We study the group E ( X ∧ m ) for the m-fold smash product X ∧ m . We show that the two obvious homomorphisms φ : S m → E ( X ∧ m ) and ψ : E ( X ) m → E ( X ∧ m ) define a homomorphism Ψ : E ( X ) m ⋊ S m → E ( X ∧ m ) for any space X, where E ( X ) m ⋊ S m is the semi-direct product of the product group E ( X ) m by the symmetric group S m . We show that in most cases the homomorphism φ : S m → E ( X ∧ m ) is a monomorphism and the kernel of Ψ is isomorphic to the kernel of ψ. The injectivity of Ψ is established for the complex projective n-space C P n ( n ≥ 2 ) , and hence, E ( ( C P n ) ∧ m ) contains a subgroup isomorphic to { ± 1 } m ⋊ S m . Sufficient conditions for Ψ to be injective are obtained for the Eilenberg–MacLane complex K ( A r , n ) where A is a subring of Q or a ring Z / k ( k ≥ 2 ) and A r is the free A-module of rank r. From this result, we see that E ( K ( A r , n ) ∧ m ) contains a subgroup isomorphic to G L r ( A ) m ⋊ S m in many cases.

In this study, DUAL measurements of finger pulse waves were taken from three children (Child A, Child B, and Child C) and their nursery teacher longitudinally (when children were 3 years old and 4 years old). Behaviors of difficult children were evaluated and scored by using "Checklist for Difficult Children" (S. Shimano, 2014) [1]. Four subscales of "troubles (anti-social behaviors)," "dissocial behaviors," "autism behaviors," and "hyperactive behaviors" were evaluated with "Checklist for Difficult Children." Three children who exhibited high scores for all four subscales were selected for the study. According to the nursery teacher, Child A was diagnosed with autism spectrum disorder. Child B's mother had an attitude problem in childrearing, so the child was not disciplined by her. And Child C showed signs of attachment disorder. DUAL measurements were taken from Child A, Child B, and Child C, respectively paired with their nursery teacher, to visualize any mental interactions with the teacher.

The fundamental theorem on coalgebras asserts that coalgebras are locally finite in the case where the ground ring is a field. We prove the local finiteness theorem of corings under the semihereditarity condition on the base algebra and the projectivity condition on a coring. This result generalizes not only the fundamental theorem on coalgebras but also Hazewinkel’s result on the local finiteness of coalgebras over a principal ideal domain and Bergman’s unpublished result on the local finiteness of corings over a semisimple Artinian ring.

We introduce a new approach to phantom maps which largely extends the rational-ization-completion approach developed by Meier and Zabrodsky. Our approach enables us to deal with the set Ph(X,Y) of homotopy classes of phantom maps and the subset SPh(X,Y) of homotopy classes of special phantom maps simultaneously. We give a sufficient condition for Ph(X,Y) and SPh(X,Y) to have natural group structures, which is much weaker than the conditions obtained by Meier and McGibbon. Previous calculations of Ph(X,Y) have generally assumed that [X,ΩŶ] is trivial, in which case generalizations of Miller’s theorem are directly applicable, and calculations of SPh(X,Y) have rarely been reported. Here, we calculate not only Ph(X,Y) but also SPh(X,Y) in many important cases of nontrivial [X,ΩŶ].

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.

Let ACG be the category of cogroups in the category A of connected graded algebras over a fixed commutative ring R. We study the full subcategory ACGco consisting of objects whose underlying algebras are graded commutative, together with the full subcategory AcoCG consisting of cocommutative objects and the full subcategory ACGco consisting of objects whose underlying coalgebras are graded cocommutative. We establish categorical equivalences of these full subcategories with categories of simpler algebraic objects, and obtain the inclusion relation of the full subcategories. Since H⁎(ΩX;R) is a cogroup in A for a 1-connected co-H-group (under the assumption that R is a field), the algebraic results are applied to the theory of co-H-groups. We study when H⁎(ΩX;R) is in AcoCG or ACGco, and generalize a theorem of Kachi.

A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplankton Synechococcus sp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells of Synechococcus sp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps.

The Lon AAA+ protease degrades damaged or misfolded proteins in its intramolecular chamber. Its activity must be precisely controlled, but the mechanism by which Lon is regulated in response to different environments is not known. Facultative anaerobes in the Enterobacteriaceae family, mostly symbionts and pathogens, encounter both anaerobic and aerobic environments inside and outside the host's body, respectively. The bacteria characteristically have two cysteine residues on the Lon protease (P) domain surface that unusually form a disulfide bond. Here we show that the cysteine residues act as a redox switch of Lon. Upon disulfide bond reduction, the exit pore of the P-domain ring narrows by ∼30%, thus interrupting product passage and decreasing activity by 80%; disulfide bonding by oxidation restores the pore size and activity. The redox switch (E°' = -227 mV) is appropriately tuned to respond to variation between anaerobic and aerobic conditions, thus optimizing the cellular proteolysis level for each environment.

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.

Sirtuin4 (Sirt4) is one of the mammalian homologues of Silent information regulator 2 (Sir2), which promotes the longevity of yeast, C. elegans, fruit flies and mice. Sirt4 is localized in the mitochondria, where it contributes to preventing the development of cancers and ischemic heart disease through regulating energy metabolism. The ADP-ribosylation of glutamate dehydrogenase (GDH), which is catalyzed by Sirt4, downregulates the TCA cycle. However, this reaction mechanism is obscure, because the structure of Sirt4 is unknown. We here constructed structural models of Sirt4 by homology modeling and threading, and docked nicotinamide adenine dinucleotide

The nature of renal amyloidosis involving Bence-Jones proteins in multiple myeloma is still unclear. The development of amyloidosis in neurodegenerative diseases is often associated with a high content of asparagine and glutamine residues in proteins forming amyloid deposits. To estimate the influence of Asn and Gln residues on the aggregation of Bence-Jones protein BIF, we obtained recombinant BIF and its mutants with the substitution of Tyr187&rarr, Asn (Y187N) in &alpha, helix of CL domain, Lys170&rarr, Asn (K170N) and Ser157&rarr, Gln (S157Q) in CL domain loops, Arg109&rarr, Asn in VL-CL linker (R109N) and Asp29&rarr, Gln in VL domain loop (D29Q). The morphology of protein aggregates was studied at pH corresponding to the conditions in bloodstream (pH 7.2), distal (pH 6.5) and proximal renal tubules (pH 4.5) by atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The Lys170&rarr, Asn replacement almost completely inhibits amyloidogenic activity. The Y187N forms fibril-like aggregates at all pH values. The Arg109&rarr, Asn replacement resulted in formation of fibril-like structures at pH 7.2 and 6.5 while the substitutions by Gln provoked formation of those structures only at pH 7.2. Therefore, the amyloidogenic properties are highly dependent on the location of Asn or Gln.

Takeuchi asserted that if a bialgebra [Formula: see text] over a field [Formula: see text] is finitely generated as a [Formula: see text]-algebra, then [Formula: see text] is a matric bialgebra. We introduce the notion of a matric coalgebra over a commutative ring [Formula: see text]. We show that if [Formula: see text] is faithfully projective as a [Formula: see text]-module, then [Formula: see text] is a matric coalgebra. Using this, we also show that if a bialgebra [Formula: see text] over a semihereditary ring [Formula: see text] is projective as a [Formula: see text]-module, then any finite subset of [Formula: see text] is contained in some matric subbialgebra. This result is a generalization of Takeuchi’s assertion and can be regarded as a local finiteness theorem on bialgebras.

A method for the determination of filamentous cyanobacteria producing musty-odor substances quickly and accurately by soft X-ray microscopy (XM) was developed and applied to Phormidum tenue sp. (Pseudanabaena sp.) green strain (PTG). To improve the identification accuracy of PTG, PTG was observed by soft XM using two wavelengths above and below the K-edge of oxygen. Two types of granules were confirmed. Since a granule of which oxygen is a major constituent element is identified as polyphosphate granule, polyphosphate granule and carboxysome are identified, respectively.

Association of protein molecules in human tissues underlies many diseases, which brings it in the focus of numerous studies. Previous reports described amyloid formation with the unfolded protein state taken as the starting point. Here, we present kinetics of protein aggregation starting from the protein native state and with consideration of its structural stability. We constructed mutant forms of apomyoglobin (apoMb) and investigated them by light scattering, small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), atomic force microscopy (AFM), fluorescence, far UV CD and FTIR spectroscopy. We found that apoMb mutants formed aggregates that changed their shape after 24 h incubation at a physiologically relevant temperature of 40 °C, pH 5.5, from almost globular (micellar) to elongated and curly. The elongated particles exhibited properties of the cross-beta structure characteristic of amyloids. According to SAXS, the elongated aggregates were not less than 300 A in length, the gyration radius of their cross-section (Rc) was about 35 A, and their molecular mass was over 400 kDa. An elongated shape of the aggregates was also demonstrated by TEM- and AFM imaging. The ability of apoMb mutants to form elongated amyloid-like aggregates, as well as their compactness, negatively correlated with stability of their native structure.

A statistical mechanical theory on the effects of the denaturant molecule on the protein helix–coil transition is developed. The unfolding agents are assumed to attach to the polypeptide backbones and disturb the formation of the hydrogen bond. The polypeptide becomes denaturated by the following reaction: h (helix) ⇌c (random coil) ⇌c* (random coil attached by denaturant). The results obtained from the theory are compared with the experimental result of short oligopeptides.

We investigated refolding processes of beta-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 degrees C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable alpha-helical burst at -15 degrees C and -20 degrees C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at -20 degrees C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of alpha-helical content at the same temperature. In case of Ki67 FHA domain, ellipticity change at 222 nm was observed on its refolding pathway at -28 degrees C in the presence of 45% EGOH and 2 mM DTT, indicating that Ki67 FHA domain also takes non-native alpha-helix-rich intermediate on its folding pathway. Time-resolved SAXS experiment was done. As the signal/noise ratio is low, we could not observe the time-dependent signal change through the time course. However, the initial Rg value was obtained as 18.2 +/- 0.5 A, which is much smaller than the unfolded Rg value (26.5 +/- 1.2 A), and is slightly larger than the native one (15.9 +/- 1.8 A). These results suggest that Ki67 FHA domain also forms compact non-native alpha-helix-rich intermediate before refolding to its native beta-structure on the refolding pathway. These results are in good agreement with other beta-proteins, such as bovine beta-lactoglobulin (BLG), src SH3 domain proteins. It seems the alpha-helical burst phases appear on the folding pathway of beta-sandwiched proteins.

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.

Understanding of macroalgal dispersal has been hindered by the difficulty in identifying propagules. Different carrageenans typically occur in gametophytes and tetrasporophytes of the red algal family Gigartinaceae, and we may expect that carpospores and tetraspores also differ in composition of carrageenans. Using Fourier transform infrared (FT-IR) microspectroscopy, we tested the model that differences in carrageenans and other cellular constituents between nuclear phases should allow us to discriminate carpospores and tetraspores of Chondrus verrucosus Mikami. Spectral data suggest that carposporophytes isolated from the pericarp and female gametophytes contained κ-carrageenan, whereas tetrasporophytes contained λ-carrageenan. However, both carpospores and tetraspores exhibited absorbances in wave bands characteristic of κ-, ι-, and λ-carrageenans. Carpospores contained more proteins and may be more photosynthetically active than tetraspores, which contained more lipid reserves. We draw analogies to planktotrophic and lecithotrophic larvae. These differences in cellular chemistry allowed reliable discrimination of spores, but pretreatment of spectral data affected the accuracy of classification. The best classification of spores was achieved with extended multiplicative signal correction (EMSC) pretreatment using partial least squares discrimination analysis, with correct classification of 86% of carpospores and 83% of tetraspores. Classification may be further improved by using synchrotron FT-IR microspectroscopy because of its inherently higher signal-to-noise ratio compared with microspectroscopy using conventional sources of IR. This study demonstrates that FT-IR microspectroscopy and bioinformatics are useful tools to advance our understanding of algal dispersal ecology through discrimination of morphologically similar propagules both within and potentially between species.

X-ray fluorescence (XRF) microscopy features unique capabilities which make it well suited for biological investigations. Its high sensitivity together with high spatial resolution and penetration depth provide a unique tool for trace elements analysis in heterogeneous samples. Like most of the X-ray based techniques, radiation damage sets hard limits on the ultimate performance. Although the interactions between matter and photons are well described from a physics point-of-view, there is a lack of experimental data, in particular for XRF imaging mode. In this context, this work proposes a practical approach in addressing the limits set by radiation damage to X-ray fluorescence imaging in the case of hydrated and unfixed cells at room temperature. We find that the maximum dose tolerated by ascidian blood cells is 105 Gy. A simple theoretical model allowed the minimal doses required for a good image contrast to be determined for various experimental schemes. The results are consistent with the experimental observation on ascidian blood cells which exemplifies the peculiar case of highly concentrated samples (>10,000 ppm) at room temperature. The same simple model predicts that in the case of the detection of high Z trace elements in cryo-preserved cells, the relative detection limit set by radiation damage is below 0.1 ppm at a spatial resolution of 100 nm.

HIV-1 protease is responsible for the maturation of infective virions, and is one of the targets of drugs against AIDS. It is an aspartic protease with a 99-resiude polypeptide dimerized. Previous study with fluorescence and sedimentation measurements revealed that the protein was unfolded with concomitant dissociation of the subunits. In the present study, we investigated urea-dependent unfolding of HIV-1 protease with CD and SAXS in order to monitor the secondary structure and the global size and shape of the molecule, respectively. The unfolding parameters estimated by both methods were almost the same, indicating that the dissociation of the subunits accompanied the disruption of their internal structures. This is in line with the previous results, and moreover some residual structures were suggested to be present in the unfolded state. The distinct difference, as compared with the unfolding of pepsin, was interpreted from the point of their molecular architectures.

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

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.

Recently, we have found a transient intermediate on the folding pathway of src SH3. Intending to investigate the structure of the transient intermediate, we tested a mutant of src SH3, named A45G, using circular dichroism, fluorescence and X-ray solution scattering, and incidentally found that it forms a stable alpha-helix-rich intermediate (I(eq)) (different from the native beta-sheet-based secondary structure) at pH 3.0, but contains only beta-sheets at pH 6.0, whereas wild-type SH3 forms only beta-sheets at both pH 3.0 and pH 6.0. The intermediate I(eq) shows a circular dichroism measured at theta(222)=-10,300 deg.cm(2) dmol(-1), indicating a 31% alpha-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I(eq) as 19.1 A at pH 3.0 and 15.4 A at pH 6.0, and Kratky plots showed a clear peak at pH 3.0, 4.0 and 6.0, indicating that I(eq) too is compact. In these parameters, I(eq) closely resembles the kinetically-obtained intermediate I(kin) which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 A and theta(222)=-8700 deg.cm(2)dmol(-1)), indicating a 26% alpha-helix proportion in our previous paper. Refolding experiments with A45G were done at pH 6.0 by stopped-flow apparatus monitored by circular dichroism, and compared to kinetic experiments with wild-type SH3 at pH 6.0. The result showed an alpha-helix-rich intermediate at the same dichroism amplitude, but nine times slower in formation-rate. A pH-jump experiment from pH 3.0 to pH 5.9 on A45G was also performed. This showed no bursts, and the rate of conformation-change was almost as fast as the refolding rate of A45G at pH 6.0. These kinetic experiment data would be consistent with I(eq) being nearly identical to the I(kin), which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3.

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 A, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D 20,w = (2.7 ± 0.1)·10−7 versus (5.8–6.0)·10−7 cm2 s−1), confirming protein association under equilibrium 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.

The lambda(6-85)(*) pseudo-wild type of lambda repressor fragment is a fast two-state folder (k(f) approximately 35 microsec(-1) at 58 degrees C). Previously, highly stable lambda(6-85)(*) mutants with k(f)30 microsec(-1) have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune lambda(6-85)(*) away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of lambda(6-85)(*) in a stabilizing solvent, 45% by weight aqueous ethylene glycol at -28 degrees C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X-ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low-barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two-state folding of lambda(6-85)(*) is not a robust process.

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.

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.

The unfolding kinetics of creatine kinase (CK) in various concentrations of urea or guanidine hydrochloride (GuHCl) was investigated by small angle X-ray scattering (SAXS) using synchrotron radiation, and compared with the results obtained by stopped-flow circular dichroism and stopped-flow fluorescence. Using the three methods, the unfolding kinetics of CK fits well to a single exponential function with similar apparent rate constants, and the amplitude of the monophasic kinetics covers the entire range of the equilibrium values. The results suggest that the unfolding time-course measured by integrated SAXS intensity corresponds to the intramolecular loss of globular structure. The refolding kinetics of 8 M urea-denatured CK was monitored in a stopped-flow apparatus by following the spectroscopic changes, and the final state of folding was investigated by SAXS. A substantial part of the ellipticity is recovered within a burst phase, indicating that the secondary structure forms at an early stage in refolding. The R(g) value of the final folded state was 33.6 A when the folding buffer contained 20% glycerol, which is characteristic of native-like compactness and globularity.

A new algorithm to refine protein structures in solution from small-angle X-ray scattering (SAXS) data was developed based on restrained molecular dynamics (MD). In the method, the sum of squared differences between calculated and observed SAXS intensities was used as a constraint energy function, and the calculation was started from given atomic coordinates, such as those of the crystal. In order to reduce the contribution of the hydration effect to the deviation from the experimental (objective) curve during the dynamics, and purely as an estimate of the efficiency of the algorithm, the calculation was first performed assuming the SAXS curve corresponding to the crystal structure as the objective curve. Next, the calculation was carried out with `real' experimental data, which yielded a structure that satisfied the experimental SAXS curve well. The SAXS data for ribonuclease T1, a single-chain globular protein, were used for the calculation, along with its crystal structure. The results showed that the present algorithm was very effective in the refinement and adjustment of the initial structure so that it could satisfy the objective SAXS data.

The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants D15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP). The native state is globular and predominantly dimeric prior to the onset of unfolding. Rg values of 32 and 45 A were obtained for the native and 5 M GdmCl denatured states of D15-42Ure2, respectively; the corresponding values for 90Ure2 were 2–3 A lower. SAXS suggests residual structure in the 4 M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions. Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions. The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation. 2003 Elsevier Inc. All rights reserved.

pH-induced unfolding process of the acid proteinase aspergillopepsin II was studied kinetically by stopped-flow methods using circular dichroism and small angle X-ray scattering. Native aspergillopepsin II consists of two polypeptide chains, a light chain and a heavy chain, which are bound noncovalently to each other at acidic pH. Above neutral pH the two chains are known to dissociate and swell due to electrostatic repulsion inside the molecule. The present studies suggested that the polypeptide chains dissociate first and that the dissociated heavy chain expands subsequently. This two-phased pathway is consistent with the accumulation of an intermediate species in the pH-induced unfolding of the enzyme.

The fluorescent Ub* mutant of the small globular protein ubiquitin is studied by multiple spectroscopic techniques under low temperature/high viscosity conditions to reveal unambiguously the formation of a compact structured intermediate preceding the barrier crossing to the native state. When detected by fluorescence intensity alone, ubiquitin appears to fold via a quasi two-state mechanism. In contrast, circular dichroism reveals the early formation of a structured ensemble, whose signal between 210 and 230 nm exceeds that of the native state, and whose unfolding curve is cooperative. The ensemble radius of gyration determined by small-angle X-ray scattering is only 1.2 times that of the native state, close to the classical molten globule value. Experiments performed in water/ethylene glycol mixtures at temperatures as low as −20 °C allow us to set an upper limit of ≤8kBT on the barrier height for formation of the intermediate ensemble. The very small fluorescence burst phase indicates that the tryptoph...

In order to improve the low signal-to-noise ratio of the time-resolved small-angle X-ray scattering, we have used a two-dimensional X-ray detector with a beryllium-windowed X-ray image intensifier and a charge-coupled device as an image sensor, and applied this to studies on (1) the kinetic folding reaction of α-lactalbumin, which accumulates the molten globule-like intermediate at an early stage of refolding and (2) the cooperative conformational transition ofEscherichia colichaperonin GroEL induced by ATP, which occurs in an allosteric manner between the close and open conformational states. In the α-lactalbumin reaction, we have firmly established the equivalence between the kinetic intermediate and the equilibrium molten globule state, and obtained further information about dehydration from the highly hydrated folding intermediate during a late stage of refolding. In the chaperonin study, we have successfully observed the kinetics of the allosteric transition of GroEL that occurs with a rate constant of about 3–4 s−1at 5°C. The combination of the time-resolved X-ray scattering with other spectroscopic techniques such as circular dichroism and intrinsic fluorescence is thus very effective in understanding the conformational transitions of proteins and protein complexes.

Solution small-angle X-ray scattering (SAXS) is an effective technique for quantitatively measuring the compactness and shape of proteins. We use SAXS to study the structural characteristics and unfolding transitions induced by urea for full length Escherichia coli trigger factor (TF) and a series of truncation mutants, obtaining and comparing the radiuses of gyration (Rg), the distance-distribution function (P(r) function) and integrated intensity of TF variants in native and unfolding states. The C-terminal 72-residue truncated mutant TF360 exhibited dramatic structural differences and reduced stability compared with the whole TF molecule, while the N-domain truncated mutant MC maintained its compact structure with reduced stability. These results indicate that the C-terminal region of TF plays an important role in the structural and conformational stabilities of the TF molecule, while the N-domain is relatively independent.

A hard X-ray microbeam with submicrometer spot size from third-generation high brilliance synchrotron radiation (SR) sources is expected to be a powerful tool for various fields of research. A Fresnel zone plate (FZP) is one of the promising, focusing elements for X-rays. Focusing efficiency is one of the important features of the FZP. In order to fabricate an FZP with a high focusing efficiency, it is necessary to investigate the relation between the experimental values of the focusing efficiency and the theoretical ones. We have fabricated Ag/C and Cu/Al multilayer (sputtered-sliced) FZPs for use in hard X-ray region, and have compared the measured focusing efficiency data with the calculated ones. The experimental data have been in good agreement with the theoretical values.

[2,3]-Wittig rearrangement of 1,1,2-trifluoroallylic ethers gave five types of novel 4,4,5-trifluroalk-1,5-dien-3-ols. The rearrangement reaction gave the alcohols with perfect (E)*-selection over the newly created olefin bond for two substrates. Lipase-catalyzed optical resolution of 4,4,5-trifluoroalk-1,5-dien-3-ols was successfully performed to afford optically active partly gem-difluorinated allylic alcohols for the first time.

Aspergillopepsin II (EC 3.4.23.6) secreted from the fungus Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase. It consists of two polypeptide chains (i.e., a heavy chain and a light chain), which are bound noncovalently to each other. The pH titration analysis using small-angle X-ray scattering (SAXS) as well as circular dichroism (CD) and gel filtration indicated that the enzyme was unfolded around a neutral pH with concomitant dissociation of the two chains. Detailed analyses showed that the midpoint pH values for the unfolding are not coincident with one another (pH 6.1 in circular dichroism and gel filtration, pH 6.4 in zero-angle intensity of SAXS, pH 6.8 in radius of gyration). The difference between these values suggested the existence of an intermediate state during the unfolding. Further analyses of the SAXS data showed that the heavy chain just after the dissociation still kept molecular compactness and that it gradually increased its dimensions as the pH was further raised. Noncoincidence of the two phenomena (i.e., chain dissociation and swelling) led to elucidation of a novel intermediate state during unfolding, which was confirmed by the subsequent singular value decomposition (SVD) analysis.