Viruses, protein separation

One of the most intriguing recent examples of disordered structure is in tomato bushy stunt virus (Harrison et ah, 1978), where at least 33 N-terminal residues from subunit types A and B, and probably an additional 50 or 60 N-terminal residues from all three subunit types (as judged from the molecular weight), project into the central cavity of the virus particle and are completely invisible in the electron density map, as is the RNA inside. Neutron scattering (Chauvin et ah, 1978) shows an inner shell of protein separated from the main coat by a 30-A shell containing mainly RNA. The most likely presumption is that the N-terminal arms interact with the RNA, probably in a quite definite local conformation, but that they are flexibly hinged and can take up many different orientations relative to the 180 subunits forming the outer shell of the virus particle. The disorder of the arms is a necessary condition for their specific interaction with the RNA, which cannot pack with the icosahedral symmetry of the protein coat subunits. 

Many protein molecules are composed of more than one subunit, where each subunit is a separate polypeptide chain and can form a stable folded structure by itself. The amino acid sequences can either be identical for each subunit (as in tobacco mosaic virus protein), or similar (as in the a and )3 chains of hemoglobin), or completely different (as in aspartate transcarbamylase). The assembly of many identical subunits provides a very efficient way of constructing... 

Poorly soluble virus proteins have been separated by RPC through gradients of formic acid/acetonitrile. Even under these harsh conditions about 100% recovery was found 71 K... 

A continuous Fl-FFF channel analogous to a dialysis cell or ultrafiltration cell [247] was described theoretically and later demonstrated in practice for the separation of bovine serum albumin from methylene blue, various viruses, proteins, and colloidal silica particles. 

Urea affects the gel as well as the state of aggregation of solutes. Stepa-now et al. (1961) have shown that formation of peptide-peptide complexes may be avoided in phenolate or alkaline urea solutions. Two peptides derived from tobacco mosaic virus protein could be separated with Sephadex only in the presence of 8 M urea. Urea need only be included in the sample solution, not in the eluting solvent (0.01 M sodium hydroxide). Upon filtration on a Sephadex G-50 column, urea and the two peptides moved as well-separated zones. The authors did not comment on the choice of G-.50. It was probably found to be superior to G-25 since urea seems to close the pores and meshes of a Sephadex gel. The reduction in effective pore and mesh size is perhaps caused by urea being bound to the carbohydrate network since the swelling is in fact increased in strong urea solutions. 

It is interesting that the presence or absence of poly(c) coincides with the classification of enteroviruses, cardioviruses, rhinoviruses and PM) viruses as separate genera, proposed in (4). Frisby (l 5) has suggested that this implies a non-involvement of the poly(c) tract in a universal function such as replication, and that it might have some specific role in assembly, as a nucleation site, interacting with the capsid proteins of those viruses which contain it. The capacity of poly(C) to form double-stranded helices at pH 5 7 or below mi t contribute to the known instability of cardioviruses and FM) viruses at acid pH, which has been employed as a criterion in the classification of the picornavirus genera (4) ... 

In contrast, the virus proteins are readily solubilized in either guanidine hydrochloride or SDS containing buffers. Figures 3A and B demonstrate the protein separation obtained when Identical samples of an equine retrovirus were dissolved and chromatographed In guanidine hydrochloride and SDS, respectively. These chromatograms demonstrate that either... 

The PDB is the repository for solved 3-D structures of proteins, peptides, viruses, protein-nucleic acid complexes, nucleic acids, and carbohydrates. Most structures deposited in the PDB are based on X-ray crystallography (approximately 80%) with the remaining structures solved using nuclear magnetic resonance (NMR) techniques. The PDB no longer stores theoretical protein models in the main archive, but it does accept them for storage in a separate theoretical model section where they are neither aimotated nor validated. The PDB staff members annotate and validate the submitted 3-D structures to ensure the information provided to the public is correct. The structures archived at the PDB are provided in two formats, PDB and... 

Figure 6 RPC of murine leukemia virus proteins. Rve milligrams of virus disrupted with guanidine HCI was injected onto a //-Bondapak phenyl column. Separation was achieved at pH 2.0 (TFA) with linear gradients of acetonitrile (- - ) at 23 =C and 1 -propanol (...) at 50°C at a flow rate of 0.2 mL/min. Absorbance was monitored at 206 nm. Shaded areas were used to determine molar ratios of proteins in mature virus. (From Ref. 38.)...

Soluble recombinant protein extracted fiom cells or supernatants of eukaryotic cells (e.g, Chinese hamster ovary cells or insea cells expressing recombinant baculovlrus) or baaeria (such as . colt harbouring plasmids or recombinant viruses) and dissolved in PBS Protein separated electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels (SDS-PAGE) and eluted from gel slices into PBS Cultured cells grown in suspension or as monolayers in flasks, then detached by treatment with 0,1% trypsin-EDTA, and resuspended in PBS or serum-free DMEM... 

RP chromatography has been used to separate a wide range of proteins of many types, but much of the published work has been concerned with a few restricted classes growth factors, peptide hormones and immunomodulators, virus proteins, haemoglobins and ribosomal proteins account for half of published separations. It is no coincidence that, in most of these cases, the proteins were either structurally robust or the retention of biological activity was of secondary importance. 

Alphaviruses, such as Sindbis virus and Semliki Forest virus, are a group of mosquito-borne, enveloped RNA viruses that can cause encephalitis, fever, arthritis and rashes in mammals. These viruses have two protein shells—an outer glycoprotein layer and an inner core— which are separated by a lipid bilayer, a membrane. Studies by cryoelectron microscopy have shown that... 

Figure 16.21 Structure of one subunit of the core protein of Slndbls virus. The protein has a similar fold to chymotrypsin and other serine proteases, comprising two Greek key motifs separated by an active site cleft. The C-terminus of the protein is bound in the catalytic site, making the coat protein inactive (Adapted from S. Lee et al., Structure 4 531-541, 1996.)...

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Western blot A method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. Some diagnostic applications for the Western blot include Lyme disease, bovine spongiform encephalopathy, and human immunodeficiency virus (HIV) (it is considered the gold standard for HIV diagnostic testing). 

Complex viruses Some virions are even more complex, being composed of several separate parts, with separate shapes and symmetries. The most complicated viruses in terms of structure are some of the bacterial viruses, which possess not only icosahedral heads but helical tails. In some bacterial viruses, such as the T4 virus of Escherichia coli, the tail itself is a complex structure. For instance, T4 has almost 20 separate proteins in the tail, and the T4 head has several more proteins. In such complex viruses, assembly is also complex. For instance, in T4 the complete tail is formed as a subassembly, and then the tail is added to the DNA-containing head. Finally, tail fibers formed from another protein are added to make the mature, infectious virus particle. 

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