A synthetic resilin is largely unstructured

Proresilin is the precursor protein for resilin, an extremely elastic, hydrated, cross-linked insoluble protein found in insects. We investigated the secondary-structure distribution in solution of a synthetic proresilin (AN16), based on 16 units of the consensus proresilin repeat from Anopheles gambiae. Raman spectroscopy was used to verify that the secondary-structure distributions in cross-linked AN16 resilin and in AN16 proresilin are similar, and hence that solution techniques (such as NMR and circular dichroism) may be used to gain information about the structure of the cross-linked solid. The synthetic proresilin AN16 is an intrinsically unstructured protein, displaying under native conditions many of the characteristics normally observed in denatured proteins. There are no apparent α-helical or β-sheet features in the NMR spectra, and the majority of backbone protons and carbons exhibit chemical shifts characteristic of random-coil configurations. Relatively few peaks are observed in the nuclear Overhauser effect spectra, indicating that overall the protein is dynamic and unstructured. The radius of gyration of AN16 corresponds to the value expected for a denatured protein of similar chain length. This high degree of disorder is also consistent with observed circular dichroism and Raman spectra. The temperature dependences of the NH proton chemical shifts were also measured. Most values were indicative of protons exposed to water, although smaller dependences were observed for glycine and alanine within the Tyr-Gly-Ala-Pro sequence conserved in all resilins found to date, which is the site of dityrosine cross-link formation. This result implies that these residues are involved in hydrogen bonds, possibly to enable efficient self-association and subsequent cross-linking. The β-spiral model for elastic proteins, where the protein is itself shaped like a spring, is not supported by the results for AN16. Both the random-network elastomer model and the sliding β-turn model are consistent with the data. The results indicate a flat energy landscape for AN16, with very little energy required to switch between conformations. This ease of switching is likely to lead to the extremely low energy loss on deformation of resilin.     

Studies on the biosynthesis of TMV

Summary Heating of the TMV replicative form (RF) above a certain temperature (T _m)causes a sharp shift from RNase resistance to sensitivity. The T _mwas determined at different salt concentrations and in the presence of formamide.The kinetics of the annealing reaction between TMV RNA and its complementary RNA was studied, and the rate constant was estimated. Under the chosen conditions which are appropriate for annealing, no dissociation of double-stranded TMV RNA was detected. The kinetic data permitted a maximum estimate of the equilibrium constant of the annealing (or dissociation) reaction.     

Studies on the biosynthesis of TMV

Summary RNA synthesis was investigated at different stages of systemic infections in which TMV formation approaches synchrony. The phenol-extracted nucleic acids were chromatographed on methylated albumin coated kieselguhr columns. At the onset of the rapid phase of virus particle formation RNA with the same chromatographic properties as TMV-RNA was predominantly synthesized when the leaves were labeled for 1.5 hours with ¹⁴C-uridine. Only a small amount of label was found in the double-stranded replicative form. At late stages of infection proportionally more replicative form was synthesized.When leaves at the start of the rapid phase of virus particle formation were labeled for 3.5 min only, a considerable amount of label was found in the replicative form. The proportion of radioactivity in this structure decreased with increasing labeling time, suggesting an intermediary function of this RNA.     

Studies on the biosynthesis of TMV

Summary The replicative form and the replicative intermediate of TMV-RNA were isolated from synchronously infected tobacco leaves, labeled with H³-uridine for 1 hour. The replicative form is over 90% resistant to RNase and sediments slightly slower than the 16S ribosomal RNA. Sucrose gradient centrifugation of the thermally denatured replicative form revealed that it contains strands of the same size as single-stranded TMV-RNA. The replicative intermediate showed only partial resistance to RNase and heterogeneous sedimentation behavior in sucrose gradients. After mild RNase treatment the replicative intermediate sedimented homogeneously, and with an S value slightly lower than the replicative form.The following abbreviations are used RF replicative form - RI replicative intermediate - STE 0.1 M NaCl-1 mM Tris-HCl-1 mM EDTA, pH 7.4 - SSC 0.15 M NaCl-0.015 M sodium citrate, pH 7.0 - 10xSSC and 0.1xSSC tenfold concentrated and tenfold diluted SSC respectively - MAK methylated albumin coated kieselguhr     

Studies on the biosynthesis of avermectins

To elucidate the pathway of avermectin biosynthesis, the biosynthetic relationships of avermectins A1a, A2a, B1a, B2a, and their respective monosaccharides and aglycones were studied. 14C-labeled avermectin compounds prepared from [1-14C]acetate were fed to Streptomyces avermitilis strain MA5502 and their metabolites were determined. Two furan ring-free aglycones, 6,8a-seco-6,8a-deoxy-5-keto avermectin B1a and B2a, have been isolated from the fermentation broth of a blocked mutant of S. avermitilis. Addition of the compounds and a semisynthetic compound, 5-keto avermectin B2a aglycone, to the fermentation medium of a second blocked mutant established that the two compounds are intermediates in the avermectin biosynthetic pathway immediately preceding avermectin aglycones.     

Studies on the biosynthesis of TMV

Summary It is shown by quantitative electron-microscopical analysis that veincleared leaves from plants systemically infected with TMV contain a majority of cells in the same stage of infection. Under defined environmental conditions the leaves which will show this synchronized virus synthesis can be determined at the time of inoculation. A standardized procedure for routine use of the system is described.The host-virus system has been used to investigate the sequential development of the virus-induced cytoplasmic differentiations and inclusion bodies. The rapid phase of virus particle formation commences at veinclearing and continues for about 40 hours. The first recognizable cytoplasmic change consists of a local branching and folding of the endoplasmic reticulum. This differentiation develops into the inclusion with diffuse, flexible rods found in late stages of infection together with the virus crystals.     

The function of resilin in beetle wings

This account shows the distribution of elastic elements in hind wings in the scarabaeid Pachnoda marginata and coccinellid Coccinella septempunctata (both Coleoptera). Occurrence of resilin, a rubber-like protein, in some mobile joints together with data on wing unfolding and flight kinematics suggest that resilin in the beetle wing has multiple functions. First, the distribution pattern of resilin in the wing correlates with the particular folding pattern of the wing. Second, our data show that resilin occurs at the places where extra elasticity is needed, for example in wing folds, to prevent material damage during repeated folding and unfolding. Third, resilin provides the wing with elasticity in order to be deformable by aerodynamic forces. This may result in elastic energy storage in the wing.     

Studies on sporopollenin biosynthesis in Tulipa anthers

Summary Extensive tracer experiments were carried out on Tulipa with the aim of determining the structure and biosynthesis of sporopollenin. The radiolabeled precursors were applied using an improved technique previously selected. The sporopollenin fraction was purified using either a gentle method — hydrolyzing enzymes (pronase, amylase, amyloglucosidase, cellulase, pectinase and lipase) and alkaline hydrolysis (method A) — or by a conventional aggressive procedure, where the material was enriched by alkaline hydrolysis and treated several days with 80% phosphoric acid (method B). The ¹⁴C-labeled precursors applied were mevalonate, glucose, acetate, malonic acid, phenylalanine, tyrosine, p-coumaric acid. Regardless of the method of enrichment, a higher level of incorporation into the sporopollenin fraction was always seen with [U-¹⁴C]-phenylalanine. The level of radioactivity found in sporopollenin labeled by phenylalanine or malonate was sufficiently high for the labeled polymer to be degraded and the products released analyzed for the first time. In the case of phenylalanine-labeled sporopollenin, the main degradation component, p-hydroxybenzoic acid, was also the most heavily labeled substance. This result was not dependent on the procedure used for sporopollenin enrichment. These findings are interpreted as meaning that phenylpropane metabolism via phenylalanine-ammonia lyase is involved in sporopollenin biosynthesis.     

Studies on the biosynthesis of neurofilament proteins

To determine whether the triplet polypeptides of neurofilaments arise by degradation of precursor, we studied the biosynthesis of neurofilament polypeptides both in vivo and in cell-free systems. Neurofilament-enriched fractions and polyribosomes were prepared from the same rabbit spinal cord homogenates. At 1 h after intracisternal administration of [34S]methionine, radiolabeled neurofilament proteins were detected in spinal cord homogenates as well as in isolated filaments. When polyribosomes from rabbit spinal cord were allowed to incorporate [35S]methionine into protein, triplet polypeptides were among the proteins labeled. Addition of spinal cord polyribosomes to rabbit reticulocyte lysates led to several cycles of translation of the spinal cord mRNA; the three neurofilament polypeptides were among the proteins synthesized in this system. The results demonstrate that the triplet polypeptides of neurofilaments are synthesized as such in the course of individual translational events and do not arise from degradation of P200 or a larger precursor.     

Studies on chlorophyll biosynthesis and other things

Our research on chlorophyll biosynthesis, over a period of approximately twenty years, has been described, emphasizing those areas in which our laboratory made significant and timely contributions. References to some of our most important articles are included. Portions of the chlorophyll biosynthetic pathway, in which our own laboratory was not involved, for example, the reduction of protochlorophyllide to chlorophyllide and the phytylation of the latter to yield chlorophyll a, have not been covered in this article. Those events which preceded my involvement with chlorophyll biosynthesis, but which contributed to the formation of my own scientific personality, are mentioned briefly in the Introduction. My non-scientific avocations have been included at the request of the reviewers and Govindjee.