ABSTRACT
Herpes simplex viruses utilize glycoproteins displayed on the viral envelope to perform a variety of functions in the viral infectious cycle. Structural and functional studies of these viral glycoproteins can benefit from biochemical, biophysical, and structural analysis of purified proteins. Here, we describe a general protocol for expression and purification of viral glycoproteins from insect cells based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains as well as the protocol for crystallization of these glycoproteins. This protocol can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of glycoproteins from HSV or other herpesviruses for biochemical and structural studies.
Subject(s)
Gene Expression , Glycoproteins , Herpesvirus 1, Human , Viral Envelope Proteins , Animals , Crystallography, X-Ray , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/isolation & purification , Herpesvirus 1, Human/chemistry , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sf9 Cells , Spodoptera , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purificationABSTRACT
A novel protein construct is presented that combines a homotrimeric, triple-stranded ß-helix as a guest to a homotrimeric foldon unit from bacteriophage T4 fibritin. The ß-helical solenoid selected is short (46 residues) and is part of a subdomain of the T4 cell-puncturing device. The resultant design is trimeric and displays greatly enhanced stability over each sub-component alone. The intended goal is a design that will enable evaluation of sequence determinants that promote in-register versus out-of-register parallel ß-sheet homotrimerization. Towards that end, the importance of a set of three buried salt-bridges was evaluated by converting them to residues otherwise consistently found throughout the natural solenoid at the same positions. The critical role of the charged residues in the salt-bridges was evident in that their elimination resulted in amyloid-like aggregation.
Subject(s)
Biochemistry/methods , Models, Molecular , Protein Structure, Secondary , Viral Proteins/ultrastructure , Amino Acid Sequence , Amyloid/chemistry , Fluorescent Dyes , Molecular Sequence DataABSTRACT
Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel ß-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells.
Subject(s)
Islet Amyloid Polypeptide/chemistry , Islet Amyloid Polypeptide/metabolism , Animals , Cattle , Cell Line, Tumor , Humans , Islet Amyloid Polypeptide/antagonists & inhibitors , Peptides/antagonists & inhibitors , Peptides/chemistry , Peptides/metabolism , Protein Structure, Secondary , Rats , Serum Albumin, Bovine/pharmacology , Surface Properties/drug effectsABSTRACT
Phosphoglycerate kinase (PGK) catalyzes a reversible phospho-transfer reaction between ATP and 3-phosphoglycerate (3-PG) that is thought to require a hinge-bending motion in the protein that brings two separate substrate-binding domains together. We have used difference infrared spectroscopy to better understand the conformational changes that are unique to the PGK-ATP-3-PG complex. Caged nucleotides (caged-ADP and caged-ATP) were used to initiate nucleotide binding to PGK or PGK-3-PG complexes. The difference spectra include those of PGK-ATP minus PGK, PGK-3-PG-ATP minus PGK-3-PG, PGK-3-PG-ADP minus PGK-3-PG, and PGK-ADP minus PGK. The resulting spectra were compared in attempts to identify bands associated with each PGK complex. In addition, complementary activity assays were performed in the presence of caged-nucleotides. While PGK activity decreased in the presence of caged-ADP, the activity was not influenced by the addition of caged-ATP. The activity assay results suggest that the caged-ADP may interact with PGK substrate binding site(s) and inhibit phospho-transfer. Therefore, additional difference infrared nucleotide exchange experiments were used to isolate the differences between ADP and ATP binding to PGK. Difference FTIR spectra obtained on PGK-nucleotide-3-PG complexes show distinct bands that may result from amino acid side chains as well as structural changes in the hinge region and/or increased interactions such as salt bridges forming between the two domains. The infrared data obtained on the active ternary complexes show evidence of changes in alpha-helix and beta-structures as well as signals consistent with Arg, Asn, His, Lys, Asp, Glu, and additional side chains that are uniquely perturbed in the active ternary complex as compared to other PGK complexes.
Subject(s)
Adenosine Triphosphate/chemistry , Glyceric Acids/chemistry , Glyceric Acids/metabolism , Phosphoglycerate Kinase/chemistry , Spectrophotometry, Infrared/methods , Adenosine Triphosphate/metabolism , Binding Sites , Models, Molecular , Phosphoglycerate Kinase/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Spectroscopy, Fourier Transform InfraredABSTRACT
Between June and September 1990, a 90-minute lecture with group discussion about the human immuno-deficiency Virus (HIV) and the acquired immunodeficiency syndrome (AIDS) was held for auxiliary staff at each of the 21 government hospitals in Jamaica. After a pilot experiment in two hospitals, a standard teaching format was used in 21 further sessions at 19 sites, including information about HIV and AIDS, how HIV is and how it is not transmitted and relevant safety precautions. Pre/post tests were given at 20 of the 21 sessions. Data were analysed from all sessions where more than two-thirds of the participants completed valid pre- and post-tests. There were 286 valid paired forms from 13 sessions (12 hospitals). In pre-tests, mean scores ranged from 31 percent to 82 percent and were > 60 percent in 12 of 13 sessions. Only 22 percent of participants indicated knowing that AIDS is a group of diseases and 21 percent identified the correct minimum exposure time required to kill HIV with bleach. Only 42 percent acknowledged that they could catch HIV from a healthy-looking carrier. Mean post-test scores were significantly higher than pre-test scores in 12 of 13 hospitals (p < 0.001 eleven times; p< 0.03 once). However, < 50 percent of persons still said they could not catch HIV from a healthy-looking carrier (AU)
Subject(s)
Humans , Male , Female , Acquired Immunodeficiency Syndrome , HIV , Health Personnel/education , Jamaica , Health Knowledge, Attitudes, PracticeABSTRACT
The prevalence of avian hematozoa in 1791 birds of 80 species and 22 families from Jamaica was determined. Species of Haemoproteus were the most common hematozoan encountered. Species of Leucocytozoon were diagnosed only in three over-wintering North American migrants and the genus was absent in native Jamaican birds. The low prevalence of infection (7.4 percent) of blood parasites in Jamaican birds was closely similar to that seen in Neotropical birds and far below that noted for the Nearctic avifauna of the same families. Prevalence in adult and young birds was virtually identical; transmission occurred primarily during the period February to April (AU)
