Search PubMed⌕ Search

Biomedical subjects

S D Black

Publications and source records attributed to S D Black.

At least 19 recordsLinked to original sources
PubMed1999

Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding.

Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.

Amino Acid Sequence↗
PubMed1999

Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.

Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.

Amino Acid Sequence↗
PubMed1998

Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites.

Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.

Amino Acid Sequence↗
PubMed1998

Blastopore formation in the animal hemisphere: functional inversion of gastrulation by centrifugation of Xenopus laevis eggs.

Eggs of Xenopus laevis and many other amphibians contain a gradient of yolk platelets along the animal-vegetal axis. Small platelets predominate in the animal hemisphere, and a boundary between medium and large yolk platelets exists near the equator. The blastopore forms at this boundary at the beginning of gastrulation, in the vegetal hemisphere. Does this boundary have a role in determination of the position of the blastopore, or does cortical information predominate? Past experiments using 1g to invert the egg showed a distinct tendency to form the blastopore in the original vegetal hemisphere. Our experiments, however, have used 20g centrifugation to achieve a more complete inversion of the yolk gradient. The blastopore formed in the original animal hemisphere in >95% of surviving gastrulae, if centrifugation was begun at normalized time 0.20-0.25 of the first cell cycle. Nearly normal larvae (DAI grades 4 and 5) form in 64% of cases (ave. DAI = 3.2). These data support the idea that the position of the blastopore depends on the position of the egg's internal contents rather than cortical determinants.

Animals↗
PubMed1997

Identification of an RNA-binding-loop in the N-terminal region of signal-recognition-particle protein SRP19.

Protein SRP19 is a 144-amino-acid polypeptide that associates intimately with the signal-recognition particle RNA (SRP RNA) and serves as an important structural and functional component of the SRP. We investigated the structure and RNA-binding activity of the human SRP19 protein by the use of comparative sequence analysis, high-stringency structure prediction, proteolytic susceptibility, and site-directed mutagenesis. SRP19 was found to consist of two distinct regions (called N-terminal and C-terminal regions) that are separated by a boundary of approximately 12-15 amino acid residues. Both regions contain an alpha-helix and several beta-strands that are connected by loops or turns. In agreement with the hypothetical model, proteolytic susceptibility demonstrated the predominant accessibility of two sites: one in a surface loop of the N-terminal region (YLNNKKTIAEGR33), and another site in the C-terminal tail at residues L129 and E133. The RNA-binding activities of mutant polypeptides with changes of conserved lysines and arginines (mutants K27Q, R33Q and R34Q) demonstrated that the proteolytically accessible loop of the N-terminal region is in direct contact with the SRP RNA. In contrast, alteration of a certain basic amino acid residues in the C-terminal region (R83, K116 and R118), as well as a deletion of four amino acid residues located at the boundary between the two regions, had no effect on the RNA-binding ability. The structural model that emerges from our data is thematically similar to that of ribosomal protein S5, the N-domain of which contains a loop motif believed to interact with double-stranded RNA. The presence of a similar structural feature in protein SRP19 has significant implications for the structure and function of the SRP19-RNA complex.

Amino Acid Sequence↗
PubMed1997

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA.

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small region that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.

Amino Acid Sequence↗
PubMed1995

Cooperative assembly of signal recognition particle RNA with protein SRP19.

Signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of secretory proteins to the lipid bilayer of the endoplasmic reticulum. SRP contains the protein SRP19, which is an important structural and functional component, believed to promote the assembly of the particle. We have purified the human SRP19 protein to homogeneity from recombinant bacteria which overexpress the polypeptide, and have studied details of the binding to SRP RNA via gel mobility shift and RNase sensitivity assays. SRP19 interacts with two SRP RNA conformers with different affinities such that the more compact RNA species is bound more avidly. Furthermore, binding was found to be highly cooperative. Binding constants and Hill coefficients were determined for several mutant SRP RNAs in which individual RNA helices were deleted. These results confirmed that both SRP RNA helices 6 and 8 are important for SRP19 binding. Enzymatic RNA structure probing of a 150-nucleotide mutant SRP RNA fragment and of the corresponding RNA-SRP19 complex showed that cooperativity may be due to protein-induced conformational changes in the large domain of the SRP RNA. Finally, SRP19 bound specifically not only to SRP RNA but also to the A-form of Escherichia coli 5S ribosomal RNA, thereby indicating structural similarities between these two RNA molecules.

Base Sequence↗
PubMed1995

Amphibian development in the virtual absence of gravity.

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate that a vertebrate can ovulate in the virtual absence of gravity and that the eggs can develop to a free-living stage.

Animals↗
PubMed1994

Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3).

The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography. Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration. Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected. The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation. Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer. However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000). HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102. Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction. The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry. Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc. in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus megaterium↗
PubMed1994

On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain.

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH. semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.

Amino Acid Sequence↗
PubMed1994

Systematic site-directed mutagenesis of protein SRP19. Identification of the residues essential for binding to signal recognition particle RNA.

Protein SRP19 is an important structural and functional constituent of the signal recognition particle (SRP) and belongs to a group of RNA binding proteins that specifically recognize certain tetranucleotide loops. Systematic site-directed mutagenesis was used to identify the amino acid residues in human SRP19 essential for interaction with SRP RNA. In our studies, three different groups of mutants were constructed, and each group covered essentially the entire sequence of the SRP19 protein in a consecutive nonoverlapping fashion. Results from 10 deletion mutants followed by the analysis of 24 mutants in which adjacent five residues were changed to pentaglycine suggested that a large portion of the protein may be required for RNA binding. Further examination of 53 mutant polypeptides in which adjacent dipeptide segments were altered showed, however, that 84 of the 144 SRP19 amino acids (58%) were not important for binding to the SRP RNA. The essential amino acids cluster in five regions which encompass most of the SRP19 sequence, with the exception of residues located at the N and C termini and a predicted internal loop. The results from the systematic site-directed mutagenesis study, when combined with protein secondary structure calculations, demonstrate that SRP19 is a precisely tooled protein which associates intimately with the SRP RNA.

Amino Acid Sequence↗
PubMed1994

Membrane topology of liver microsomal cytochrome P450 2B4 determined via monoclonal antibodies directed to the halt-transfer signal.

The membrane topology of cytochrome P450 2B4 from the endoplasmic reticulum has been studied with highly-purified liver microsomes in a site-directed immunochemical approach. Microsomes were prepared from phenobarbital-induced rabbits, and the resulting microsomal fraction was washed 6 additional times with 0.1 M pyrophosphate buffer to effect removal of significant quantities of adventitiously-bound protein. Monoclonal antibodies were prepared against residues 18-29 of P450 2B4 (Leu18-Leu-Phe-Arg-Gly-His-Pro-Lys-Ala-His-Gly-Arg29), essentially corresponding to the halt-transfer signal. This region was chosen due to its mutually-exclusive location in the two alternative membrane topology models currently tenable [Black, S.D. (1992) FASEB J.6, 680-685]. Model "A" contains a single transmembrane anchor peptide with the amino terminus projecting into the lumen of the endoplasmic reticulum, while model "B" exhibits a hairpin loop of the first approximately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer; the halt-transfer signal peptide would be located at the cytosolic surface of the membrane in model "A" or as a loop on the lumenal side of the membrane in model "B". Nine antibodies, denoted as MmAbA, MmAbC, MmAbD, MmAbF, MmAbH, MmAbI, MmAbK, MmAbL, and MmAbP, were produced, and all were identified as IgM/kappa subtypes. Western blotting demonstrated that the antibodies could readily recognize P450 2B4 in microsomes. ELISA assays showed that all of the antibodies exhibited strong binding to intact microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Detergent effects in rabbit liver microsomal UDP-glucuronosyltransferase studied by means of a continuous spectrophotometric assay with p-nitrophenol.

UDP-glucuronosyltransferase (UDPGT) has been studied with a continuous spectrophotometric assay employing UDP-glucuronic acid and p-nitrophenol as substrates. Activity is linearly dependent on the microsomal protein concentration. Male rabbit liver phenobarbital-induced microsomes exhibited a rate of 7.10 microM p-nitrophenol conjugated per minute at 37 degrees C. Addition of small amounts of Tergitol NP-10 caused an approximately 4-fold increase in conjugation activity; maximal activation was observed at 0.01% (v/v) detergent. However, inclusion of additional detergent caused significant inhibition of activity, such that 0.5% Tergitol caused the rate to fall 2.5-fold below the activity observed in the absence of detergent. Membrane solubilization was studied by light scattering. At maximal stimulation of p-nitrophenol UDPGT activity, the membrane was solubilized only approximately 17%. At the point of 50% solubilization, activity was still 91% of maximum. Complete membrane solubilization was achieved at approximately 0.2% Tergitol, and transferase activity had fallen slightly below the rate observed in the absence of detergent. Possible explanations for the unusual detergent-dependence of microsomal p-nitrophenol UDPGT activity are discussed.

Animals↗
PubMed1994

Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium.

Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme from Bacillus megaterium that is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system. We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme. B. megaterium ATCC 14581 was grown to high cell density, and P450 102 was purified rapidly and in high yield by chromatography on adenosine-2',5'-diphosphate agarose from crude cell-free extract. The cytochrome bound to the column with remarkable avidity, in contrast to the significantly weaker binding observed for NADPH-cytochrome P450 reductase. Chromatographic behavior also showed that the cytochrome bound NADP(+)-type nucleotides more tightly than any other cellular polypeptide. The purified protein was electrophoretically homogeneous and had essentially theoretical contents of FAD, FMN, and heme. Optical spectra showed the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized. Molar extinction coefficients in the oxidized, fully reduced, and ferrous carbonyl states have been determined; notable is the large soret extinction in the ferrous carbonyl state (epsilon 449 nm = 143,500 M-1 cm-1). Final preparations were active in the oxidation of a wide variety of substrates. Of the C14 alkyl compounds studied, tetradecyltrimethylammonium bromide showed the highest substrate-dependent oxidation of NADPH, followed by myristate and myristyl alcohol; however, myristate exhibited the lowest Km value. Activities were tightly coupled to NADPH oxidation (> 97%). Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflourobutyrate, and 7-ethoxycoumarin were not substrates. NADP+ titrations showed, as expected, that the coenzyme was bound very tightly, with an average Kd of 580 nM. Our preparations of P450 102 are of sufficient purity and stability that crystals of the native holoenzyme have been grown.

Bacillus megaterium↗
PubMed1993

Structure of 6-fluoro-1,2,3,4,7,12-hexahydro-7-methyl-12-methylene- benz[a]anthracene.

The X-ray analysis confirms the structure of the 12-methylene tautomer formed by the acid catalysis of 6-fluoro-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene. The central C ring is in a boat conformation, with the result that the molecule is bent about a line through atoms C(7) and C(12) with a dihedral angle of 32.5 degrees. The cyclohexene A ring is in a half-chair conformation.

Benz(a)Anthracenes↗
PubMed1992

Development of hydrophobicity parameters for prenylated proteins.

We have determined hydrophobicity parameters for the side-chains of the prenyl thioether protein modifications, farnesyl-cysteine and geranylgeranyl-cysteine. Farnesyl-Cys is somewhat more hydrophobic than palmitoyl-Cys, but geranylgeranyl-Cys is more than two log(P) units more non-polar. These post-translational modifications represent the most hydrophobic residues yet described quantitatively. Furthermore, such modifications occur at the COOH-terminus which is generally methyl esterified. Loss of the terminal negative charge and formation of the ester proceeds with the gain of an additional 2.343 log(P) units of hydrophobicity. Clearly, COOH-terminal prenylation and esterification impart sufficient potential to render the terminus membrane bound. Thus, hydrophobicity parameters presented here for the prenylated amino acyl residues extend our understanding of these important physiological derivatives and enable computational analysis of proteins thus modified.

Amino Acid Sequence↗
PubMed1992

Deployment of extracellular matrix proteins in sea urchin embryogenesis.

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi-laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle.

Animals↗