Tolerance of Arc repressor to multiple-alanine substitutions

Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.

Circularly permuted green fluorescent proteins engineered to sense Ca2+

To visualize Ca2+-dependent protein–protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named “pericam,” was fluorescent and its spectral properties changed reversibly with the amount of Ca2+, probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, “flash-pericam” became brighter with Ca2+, whereas “inverse-pericam” dimmed. On the other hand, “ratiometric-pericam” had an excitation wavelength changing in a Ca2+-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca2+ dynamics, such as Ca2+ oscillations in the cytosol and the nucleus. Ca2+ imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca2+ ions across the nuclear envelope. Then, free Ca2+ concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca2+ transients caused rapid changes in the concentration of mitochondrial Ca2+. Finally, a “split-pericam” was made by deleting the linker in the flash-pericam. The Ca2+-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B- and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b5 Reductase from Arabidopsis

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b5 reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b5, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b5 reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b5 reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b5 reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b5 (AtB5-A), whereas no Cyt b5 reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b5 with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b5 reductase and NADPH-Cyt P450 reductase can reduce Cyt b5 and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.

Primate photopigments and primate color vision.

The past 15 years have brought much progress in our understanding of several basic features of primate color vision. There has been particular success in cataloging the spectral properties of the cone photopigments found in retinas of a number of primate species and in elucidating the relationship between cone opsin genes and their photopigment products. Direct studies of color vision show that there are several modal patterns of color vision among groupings of primates: (i) Old World monkeys, apes, and humans all enjoy trichromatic color vision, although the former two groups do not seem prone to the polymorphic variations in color vision that are characteristic of people; (ii) most species of New World monkeys are highly polymorphic, with individual animals having any of several types of dichromatic or trichromatic color vision; (iii) less is known about color vision in prosimians, but evidence suggests that at least some diurnal species have dichromatic color vision; and (iv) some nocturnal primates may lack color vision completely. In many cases the photopigments and photopigment gene arrangements underlying these patterns have been revealed and, as a result, hints are emerging about the evolution of color vision among the primates.

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

A multimer model for P680, the primary electron donor of photosystem II.

We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins. Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair. We find that this model is consistent with many experimental studies of PS II. The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous. This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor. We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

In vivo assembly of rhodopsin from expressed polypeptide fragments.

Rhodopsin folding and assembly were investigated by expression of five bovine opsin gene fragments separated at points corresponding to proteolytic cleavage sites in the second or third cytoplasmic regions. The CH(1-146) and CH(147-348) gene fragments encode amino acids 1-146 and 147-348 of opsin, while the TH(1-240) and TH(241-348) gene fragments encode amino acids 1-240 and 241-348, respectively. Another gene fragment, CT(147-240), encodes amino acids 147-240. All five opsin polypeptide fragments were stably produced upon expression of the corresponding gene fragments in COS-1 cells. The singly expressed polypeptide fragments failed to form a chromophore with 11-cis-retinal, whereas coexpression of two or three complementary fragments [CH(1-146) + CH(147-348), TH(1-240) + TH(241-348), or CH(1-146) + CT(147-240) + TH(241-348)] formed pigments with spectral properties similar to wild-type rhodopsin. The NH2-terminal polypeptide in these rhodopsins showed a glycosylation pattern characteristic of wild-type COS-1 cell rhodopsin and was noncovalently associated with its complementary fragment(s). Further, the CH(1-146) + CH(147-348) rhodopsin showed substantial light-dependent activation of transducin. We conclude that the functional assembly of rhodopsin is mediated by the association of at least three protein-folding domains.

Modular organization of intrinsic connections associated with spectral tuning in cat auditory cortex

Many response properties in primary auditory cortex (AI) are segregated spatially and organized topographically as those in primary visual cortex. Intensive study has not revealed an intrinsic, anatomical organizing principle related to an AI functional topography. We used retrograde anatomic tracing and topographic physiologic mapping of acoustic response properties to reveal long-range (≥1.5 mm) convergent intrinsic horizontal connections between AI subregions with similar bandwidth and characteristic frequency selectivity. This suggests a modular organization for processing spectral bandwidth in AI.