A role of methionine 100 in facilitating PYP(M)-decay process in the photocycle of photoactive yellow protein

PYP (photoactive yellow protein) is a photoreceptor protein, which is activated upon photo-isomerization of the p-coumaric acid chromophore and is inactivated as the chromophore is thermally back-isomerized within a second (in PYP(M)-to-PYP(dark) conversion). Here we have examined the mechanism of the rapid thermal isomerization by analyzing mutant PYPs of Met100, which was previously shown to play a major role in facilitating the reaction [Devanathan, S. et al. (1998) Biochemistry 37, 11563-11568]. The mutation to Lys, Leu, Ala, or Glu decelerated the dark state recovery by one to three orders of magnitude. By evaluating temperature-dependence of the kinetics, it was found that the retardation resulted unequivocally from elevations of activation enthalpy (DeltaH( double dagger )) but not the other parameters such as activation entropy or heat capacity changes. Another effect exerted by the mutations was an up-shift of the apparent pK(a) of the chromophore [the pK(a) of a titratable group (X) that controls the pK(a) of the chromophore] in the PYP(M)-decay process. The pK(a) up-shift and the DeltaH( double dagger ) elevation show an approximately linear correlation. We, therefore, postulate that the role of Met100 is to reduce the energy barrier of the PYP(M)-decay process by an indirect interaction through X and that the process is thereby facilitated.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.     

Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.     

Light-induced unfolding of photoactive yellow protein mutant M100L

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.     

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

Rapid Screening of a Novel Arrayed Medaka (Oryzias latipes) Cosmid Library

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.