Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Conformational characterization of human angiogenin by limited proteolysis

The primary structure of angiogenin is 33% identical to that of bovine pancreatic ribonuclease (RNase), but the enzymatic activities of the two proteins differ markedly. Similarly, their susceptibilities to limited proteolysis differ as well. In contrast to RNase, angiogenin totally resists proteolysis by subtilisin. Indeed, among 16 proteases examined, only endoprotease Lys-C, trypsin, and pepsin are able to cleave angiogenin. Even with prolonged incubation, endoprotease Lys-C selectively cleaves the Lys-60-Asn-61 bond; the product retains full ribonucleolytic activity. Initially, trypsin also cleaves this same bond, but with time it causes extensive degradation. Pepsin, atpH 2, cleaves the Phe-9-Leu-10 bond, to give angiogenin (10–123), which displays 15% of the native activity toward ribosomal RNA (rRNA). The susceptibility to proteolysis and/or the sites of cleavage of angiogenin and bovine RNase differ markedly despite their structural homology. These differences are considered in terms of the amino acid sequences of the two proteins.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

Interaction of alcohol and protein deficiency on rat brain synaptosomal (Na+−K+)-ATPase

Administration of low amounts of ethanol for a prolonged period increases rat brain synaptosomal (Na⁺–K⁺)-ATPase activity, the increase being less in the protein deficient rats. The adaptive mechanism to offset the stress imposed by the continued presence of ethanol seems to be depressed by low plane of nutrition. In vivo and in vitro effects of ethanol on (Na⁺–K⁺)ATPase seems to be different.     

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle

The acetylcholine receptor (AChR) content and the autorphosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rat soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [¹²⁵I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro³²P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the³²P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the³²P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.Abbreviations ACh acetylcholine - AChR acetylcholine receptor(s) - BUTX alpha-bungarotoxin - Kd kilodalton - PAGE polyacrylamide gel electrophoresis - R-II regulatory subunit of cyclic AMP-dependent protein kinase type II - SDS sodium dodecyl sulfate     

Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle