Reconstitution of the M13 major coat protein and its transmembrane peptide segment on a DNA template.
The major coat protein (pVIII) of M13 phage is of particular interest to structure biologists since it functions in two different environments: during assembly and infection, it interacts with the bacterial lipid bilayer, but in the phage particle, it exists as a protein capsid to protect a closed circular, single-stranded DNA (ssDNA) genome. We synthesized pVIII and a 32mer peptide consisting of the transmembrane and DNA binding domains of pVIII. The 32mer peptide displays typically an alpha-helical structure in trifluroethanol or 0.2 M octylglucoside solutions similar to pVIII. Attachment of polyethylene glycol (PEG) onto the N-terminal of 32mer increased the alpha-helical content and the peptide thermal stability. The peptides were reconstituted with DNA from a detergent solution into a discrete (<200 nm diameter) nanoparticle on both linear double-stranded DNA (dsDNA) and linear ssDNA, where the linear dsDNA is used to mimic the closed circular, ssDNA in M13 phage, upon removal of the detergent. The peptide/DNA particle was an irregular and not a rod-shaped aggregate when imaged by atomic force microscopy. All three peptides underwent a structural transition from alpha-helix to beta-sheet within approximately 1 h of DNA addition to the detergent solution. There was a further decrease in alpha-helical content when the detergent was removed. The presence of anionic (such as octanoic acid) or cationic (such as 1,5-diaminopentane) molecules in the detergent mixture resulted in the retention of the peptide alpha-helical structure. Thus the interaction between the peptide and DNA in octylglucoside is driven by electrostatic forces, and peptide-peptide interactions are responsible for the transition from alpha-helix to beta-sheet conformation in pVIII and its analogues. These results suggest that the assembly process to form a rod-shaped phage is a delicate balance to maintain pVIII in an alpha-helical conformation that requires either an oriented bilayer to solubilize pVIII prior to interaction with the DNA or other phage proteins to nucleate pVIII in the alpha-helical conformation on the DNA.
Li
Weijun
W
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California at San Francisco, San Francisco, California 94143-0446, USA.
Suez
Itai
I
Szoka
Francis C
FC
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary
D036022:Capsid Proteins; D010446:Peptide Fragments; C025668:coat protein, Bacteriophage M13; D004247:DNA