Increased aggregation of C-phycocyanin produced by phenol and benzene

The effect of selected aromatic compounds on aggregates of C-phycocyanin was examined by analytic ultracentrifugation. Benzene and phenol caused an increase in aggregation. The principal change, a conversion of 6S to an 11S species, is postulated to result from the interaction of benzene and phenol with hydrophobic areas involved in protein-protein contact.The C-phycocyanin used in these studies included that extracted from Spirulina platensis, a Cyanophyta that grows in a highly alkaline environment. The pH dependence of the aggregation properties of this protein, however, was similar to that of C-phycocyanins from algae grown in normal environments. This suggests that the internal biopolymers of organisms which naturally exist at extremes of pH are somehow protected from exposure to these extremes.     

Aging of tubulin at neutral pH

The aging of calf brain tubulin in neutral solution has been investigated using the techniques of sedimentation velocity and equilibrium, microtubule assembly, fluorescence spectroscopy, circular dichroism and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that tubulin incubated at 4 °C undergoes a slow association process leading to the generation of a 9 S component. The fraction of 9 S component increases progressively with incubation time and appears to follow mono-molecular kinetics. The generation of the 9 S species is paralleled closely by inhibition of microtubule assembly and loss of colchicine-binding ability. Fluorescence spectroscopy and circular dichroism spectra indicate that tryptophan moieties are perturbed during the aggregation process and that the tubulin dimer undergoes a conformational change. There is no protein degradation up to an incubation period of 50 hours. Sedimentation equilibrium experiments in 6 m-guanidine hydrochloride, both in the presence and absence of 2-mercaptoethanol, indicate that the aggregates are stabilized by disulfide bonds and hydrophobic interactions. At periods of incubation >50 hours, the protein starts to be degraded.     

Self-association of human protein S

Protein S functions as a cofactor with activated protein C in the down-regulation of the blood coagulation cascade. In vitro studies have historically produced conflicting data with regard to the extent of various protein S activity in clotting assays which typically involve adding CaCl(2) to initiate reactions. We report here that protein S reversibly self-associates in the absence of Ca(2+). Sedimentation experiments showed a transition in sedimentation velocity from 7.2 to 4.2 S with a transition midpoint (T(m)) of 0.42 mM Ca(2+) for intact protein S. Studies of thrombin cleaved (Arg(70)) protein S revealed similar results with a transition in sedimentation velocity from 7.9 to 4.4 S with a T(m) of 0.42 mM Ca(2+). This transition is reversible with the addition of 10 mM EDTA. Sedimentation equilibrium data suggest at a minimum, a monomer-dimer-trimer association. Sedimentation velocity experiments were also performed on mixtures of protein S and prothrombin which showed no heterodimer formation in either Ca(2+) or EDTA solutions. These data suggest that previous interpretations of protein S structure and function may have been confounded by the self-associative behavior of protein S in non-Ca(2+) solutions.     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Beef liver esterase. I. Isoelectric point and molecular weight

A sample of carboxylesterase has been purified from beef liver by the method of Runnegar et al. [Biochemistry8, 2013 (1969)]. The protein isoelectric point, pI was found to be 5.5, using the isoelectric focusing method. The molecular weight was found by equilibrium sedimentation to be 5.5 × 10⁴. Sedimentation velocity combined with diffusion gave a similar value. In high protein concentrations aggregation was not detected.     

Enzymes of pentose biosynthesis. The quaternary structure and reacting form of transketolase from baker's yeast

Transketolase from baker's yeast is a dimeric enzyme with a molecular weight of 158,000 ± 4000. Sedimentation velocity and sedimentation equilibrium experiments indicate that the enzyme dissociates at low concentrations (less than 0.1 mg/ml) in the absence of the coenzyme, thiamine pyrophosphate. However, no such dissociation was detected in the presence of coenzyme. Reacting enzyme sedimentation velocity measurements showed that the reacting species of the enzyme is a dimer with an s_20,w of 7.7 S.     

Pressure-induced dissociation of aggregates of myelin proteolipid protein

Sedimentation velocity and equilibrium experiments have revealed an extremely pressure-sensitive aggregation of myelin proteolipid protein in the presence of Triton X-100, dissociation of the protein aggregate being observed at pressures that are several orders of magnitude lower than those effecting disaggregation of many other proteins. These results highlight the need to employ a range of angular velocities in sedimentation studies of intrinsic membrane protein.     

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.     

Spectroscopic study of phycocyanobilin from the cyanobacteriumSpirulina platensis

Spectroscopic characterization of phycobiliprotein (PBP) isolated fromSpirulina platensis indicated that it had a molecular formula of C₃₃H₄₀N₄O₆ with molecular ion peak at m/e 586. The PBP was rich in aliphatic and acidic amino acid residues. The specific ultra-violet absorbance, infrared transmittance, specific absorbance coefficient, nuclear magnetic resonance and mass spectral analysis revealed thatS. platensis PBP was composed mainly of C-phycocyanin and allophycocyanin.     

Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.     

Post- -globulin: isolation and physicochemical characterization

Post-γ-globulin has been purified from urine of kidney transplant patients by a procedure which includes ultrafiltration, gel filtration, and ion-exchange chromatography. The purified protein appears to be homogeneous by ultracentrifugal and gel electrophoretic criteria. The yield of the purified protein was 30–35% of the total amount of post-γ-globulin excreted in different urine samples.Electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol revealed a single band and established that the protein consists of one polypeptide unit with a molecular weight of approximately 11,500. Sedimentation velocity experiments indicated a s_{20, w} of 1.55 S. Amino acid analysis of post-γ-globulin revealed the presence of hydroxylysine and hydroxyproline. The protein is devoid of carbohydrate. Post-γ-globulin has been demonstrated in small quantities in normal urine, serum, cerebrospinal fluid, and mixed saliva; preparations derived from these fluids have been shown to be immunochemically identical.     

Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 3'-extension of 5S rRNA in Saccharomyces cerevisiae

Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3′-extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5–5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5–5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p.     

A study of the states of aggregation of alfalfa mosaic virus protein.

The states of aggregation of alfalfa mosaic virus (AMV) protein have been characterized by sedimentation velocity experiments and electron microscopy. The main association product is a spherical particle with an s value of about 30S. It is highly likely that the assembly of this particle starts with dimers of the 25000 molecular mass unit resulting in an icosahedral particle made of 30 dimers. No intermediate aggregation products have been detected. The clustering pattern of the protein in the cylindrical part of the AMV capsid favours the concept of dimers as the active assembling units.     

Single molecule characterization of α-synuclein in aggregation-prone states

α-Synuclein (αS) is an intrinsically disordered protein whose aggregation into ordered, fibrillar structures underlies the pathogenesis of Parkinson's disease. A full understanding of the factors that cause its conversion from soluble protein to insoluble aggregate requires characterization of the conformations of the monomer protein under conditions that favor aggregation. Here we use single molecule Förster resonance energy transfer to probe the structure of several aggregation-prone states of αS. Both low pH and charged molecules have been shown to accelerate the aggregation of αS and induce conformational changes in the protein. We find that at low pH, the C-terminus of αS undergoes substantial collapse, with minimal effect on the N-terminus and central region. The proximity of the N- and C-termini and the global dimensions of the protein are relatively unaffected by the C-terminal collapse. Moreover, although compact at low pH, with restricted chain motion, the structure of the C-terminus appears to be random. Low pH has a dramatically different effect on αS structure than the molecular aggregation inducers spermine and heparin. Binding of these molecules gives rise to only minor conformational changes in αS, suggesting that their mechanism of aggregation enhancement is fundamentally different from that of low pH.     

Isolation and characterization of precursors in T4 baseplate assembly the complex of gene 10 and gene 11 products

The first multi-protein precursor in the assembly of the radial arms of the T4 baseplate has been purified to homogeneity. The complex was isolated from cells infected with a mutant blocked in the subsequent step in baseplate arm assembly. The assay for this precursor exploited the fact that the complex contains the target antigen of the neutralizing antibodies found in antibaseplate serum (Berget & King, 1978).The complex is composed of gene 10 protein (M_r, 88,000) and gene 11 protein (M_r, 24,000). Analytical ultracentrifugation experiments revealed a molecular weight of 258,000 and a sedimentation coefficient of 9.3 S for the complex. The overall and single polypeptide chain molecular weights are consistent with the complex containing two gene 10 polypeptides and four gene 11 polypeptides. Visualization of the complex in the electron microscope revealed an asymmetric angular structure. The shape, together with the previous identification of gene 11 product as the tail-spike protein (Crowther et al., 1977), indicates that the complex forms the body of the spikes and vertices of the hexagonal baseplate.Using an in vitro baseplate assembly assay, it was possible to demonstrate that the complex contains both the assembly-active gene 10 and gene 11 products. Gene 11 product (from 10^? extracts) can convert 11^? particles to viable phage. However, the complex lacked this activity, indicating that it does not readily dissociate. The precursor complex could be dissociated with denaturing solvents. Upon returning to physiological conditions, both the antigenic and biological activities of the gene 11 product could be recovered. The biological activity of the gene 10 product was not regained.     

Yeast Nst1 is a novel component of P-bodies and is a specific suppressor of proteasome base assembly defects

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified negative salt tolerance 1 (Nst1), a protein that when overexpressed specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6Δ rpn14Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that colocalized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6Δ rpn14Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress. Our data indicate that Nst1 is a previously overlooked P-body component that, when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation and rescues proteasome assembly under stress conditions.     

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.     

Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.