Conformational transitions in Pf3 and their implications for the structure and assembly of filamentous bacterial viruses.

Abstract

Laser Raman and circular dichroism spectra of filamentous bacteriophage Pf3 show that its coat protein is predominantly ca-helical, similar to the subunits ofbacteriophages Pf1 and fd. Unlike Pff and fd, however, the subunits of Pf3 are converted to P-sheet structures by raising the temperature, the transition temperature depending upon phage and NaCi concentrations. On cooling, the .8 structure reverts to an a structure the same as or similar to the native structure. On further heating it converts irreversibly to a second ai-helical form different from the original one. The spectra also show that aromatic amino acid residues of Ff3 undergo dramatic changes in molecular environment during the a --f3 transition. Similar transitions are observed to take place in the filamentous bacteriophage Xf.Pf3 virus is one ofseveral filamentous viruses that have a Gramnegative bacterium as host and a covalently closed singlestranded DNA molecule packed in a long (103 nm), slender (<10 nm diameter) sheath made of several thousand copies of a principal structure protein and a few copies of one or more other proteins located at the ends. The molecular biology and structures of those that have been studied most extensively, such as the closely related fd, M13, and fl, and the less related Ift, Pfl, and Xf, are described in a recent monograph (1). PM3 has Pseudomonas aeruginosa strains bearing the RP1 plasmid as hosts (2). Its length is about 700 nm (3, 4), its mass-per-length is about 19,000 daltons/nm (4), its DNA contains about 6 kilobases (4), its principal protein subunit has molecular weight 4400 (B. Frangione, personal communication), and its x-ray diffraction patterns (W. Winter, personal communication) are more like those of Xf and Pf1 than those of fd and If (5, 6). Spectral and chemical properties ofPf3 (unpublished data) show that its DNA structure is different from the DNA structures in Pf1, Xf, and fd (7,8).We report here that native Pf3 has a high content of a-helix and that dramatic transitions in protein structure occur when the temperature is raised, provided the virus concentration is high. We have also carried out some comparative measurements on Xf, Pf1, and fd, but most of the data obtained so far are for Pf3. The transitions have been observed through changes in laser Raman spectra, especially in the conformationally sensitive amide I and amide III Raman bands. These bands are widely used to distinguish a-helix, p-sheet, and irregular regions of the polypeptide backbone (9-12). A number of other bands are used to obtain structural information about protein side groups (13) and, for nucleic acids and nucleoproteins, about the bases and the sugar-phosphate backbone (14-16). Raman studies of Pf1 and fd have shown high a-helix content in the protein subunits (11, 12) as well as abnormal tyrosines (17,18) and the absence of sugar-phosphate backbone of the classic A type (11).Some aspects of the concentration dependence of the Pf3 structure transitions have been revealed by monitoring changes in circular...

Proteins can convert to β‐sheet in single crystals

“…α‐Helical‐to‐β‐sheet transitions have been reported for bacteriophage coat protein, brought about by heating the proteins in aqueous solution (Thomas et al 1981; Thomas and Day 1981). Thus, the question arises as to whether the conditions of the Raman experiments, especially any heating effects of the laser beam, bring about the observed changes in protein conformation within the crystals.…”

Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

“…Previous publications from the laboratory of the Southeastern Massachusetts University (North Dartmouth, MA) group give full details of sample handling procedures and Raman instrumentation employed to study isotopic hydrogen exchange of inosine nucleotides (Ferreira Thomas and Ferreira, 1982) and structures of the filamentous bacterial viruses Pf3 and Pf 1 (Thomas and Day, 1981;Thomas et al, 1983). Filamentous phage were generously provided by Dr. Loren A.…”

“…The native virus structure was perturbed by heating the sample at 600C for 2 h to promote the a-to ,-structure transition. (See Thomas and Day, 1981. ) cannot be made from the unrefined Raman data because of extensive overlap of the respective amide I bands.…”

Proton‐decoupled 15N and 31P solid‐state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers

“…During phage assembly at the membrane, the major coat protein undergoes conformational transformation from an integral membrane protein to a shingle‐like capsid protein. For phage assembly, the coat protein tightly associates with the single‐stranded DNA and adopts a nearly complete and extended α‐helical structure [6]. Finally, during the infection process, the coat proteins disperse from the phage into the plasma membrane, thereby releasing the DNA into the cytoplasm [7].…”