A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R g , and hydrodynamic radius, R H , of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R g and R H measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D 6 barrel" cage assembly measured by cryoelectron microscopy.A major component of the protein coats of certain endocytic vesicles is clathrin, a heteropolymer composed of a 192 kDa heavy chain and a variable, ca. 23-27 kDa, associated light chain (1). Three clathrin molecules join at a common hub to form a threelegged "triskelion", which is the basic building block of the coats. Each leg is approximately 3.0 nm thick and 52.0 nm in length and ends in a globular "terminal domain" of radius 5.0 nm. By convention, a leg is divided into a proximal segment adjoining the central hub, a linker region, and a distal segment that connects with the terminal domain. A clathrin heavy chain runs the entire length of the leg, with a light chain attached near the common hub (see Figure 1). In vitro, the triskelia assemble into polyhedral cages (or "baskets") composed of pentagonal and hexagonal faces (2), mimicking the structures seen on the outside of endocytic vesicles. One can dissociate baskets or uncoat vesicles by changing the properties † This work was supported by extramural grants from the National Institutes of Health and intramural funds from the National Institute
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Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript of the solution in which they are suspended (e.g., by changing pH and ionic strength) (3) or by adding an uncoating ATPase (4).Triskelia assemble at low pH to form baskets (3) or at higher pH, when clathrin assembly proteins (e.g., AP-2, AP-180, etc.) are added (5). It is not currently known why these conditions favor assembly. Possible mechanisms supporting assembly at low pH include conformational changes in the tertiary structure of the triskelia, which might relieve steric hindrances to basket assembly, and enhanced interactions between the intertwined triskelia that form the basket struts. The role of APs as assembly proteins may be to increase mechanical linkages between the triskelia (5-7).In this paper, we report light-scattering measurements on clathrin in solution, including conditions where assembly occurs. From static light scatteri...