Extended circular dichroism measurements using synchrotron radiation show that the assembly of clathrin coats requires no change in secondary structure.
A number of models have been proposed for the assembly of clathrin triskelia into coats. However, little is known of the effects of assembly on triskelion structure. A more detailed knowledge of the way in which assembly affects triskelion structure would be valuable for assessing the relative merits of the proposed models. The development of a vacuum-ultraviolet circular dichroism (CD) instrument that uses synchrotron radiation as a light source has allowed us to extend the range of CD measurements to shorter wavelengths. This has greatly increased signal quality even for highly scattering samples. Also, we have improved CD data analysis to provide standard deviations for calculated secondary structure content. These developments have increased the precision of CD analysis beyond what has been thus far possible. Using these developments, we have determined the secondary structure content of all components of coat protein, under both assembly and dissociating conditions. The assembly of coats does not incur any change in secondary structure content, but a 10% loss of triskelion helical content accompanies assembly in the absence of AP-2. We conclude that coat assembly requires no detectable reorganization of triskelion structure. Our result indicates that AP-2 stabilizes helical structure in the triskelion, and we propose that this increases triskelion rigidity, restricting the range of coat sizes.
Clarke
D T
DT
Synchrotron Radiation Department, CLRC Daresbury Laboratory, Warrington, UK.
Jones
G R
GR
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001923:Brain Chemistry; D002942:Circular Dichroism; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013552:Swine; D017356:Synchrotrons; D014965:X-Rays
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins