Field Guide to Gels of the assembled Forms of the HK97 major capsid protein
HK97 Proheads and Heads assayed on Native and Denaturing Gels
Purified HK97 structures
run in an agarose gel
The same set of samples used for 3 diferent gel assays.
Wild Type
(K196Y)
A
B
C
D
6
5
4
6
5
4
2
Head II
HI
gp5
42kDa
Prohead I
gp5*
31kDa
gp4
25kDa
SDS polyacrylamide gel
after SDS - incubation
Agarose native gel
of whole structure
SDS polyacrylamide gel
after TCA precipitation
HK97 capsid proteins in native (non-denaturing) gels. (A, B) The HK97 major capsid protein has not been detected as a solu-
ble monomer, but it does form a variety of higher order assemblies that can be visualized as bands in gels. Agarose gels are a
convenient way of discovering which assemblies are made by different mutants in vivo, or are produced by different treatments in
vitro. In agarose gels, fully assembled Prohead I (PI) and Prohead II (PII) make distinct bands that run at nearly the same position
(PII is slightly faster). Head II (HII), the expanded and mature HK97 capsid, runs slower than proheads in agarose gels, but so do
capsomers, which are the mixture of hexamers and pentamers made by dissociating Prohead I. Capsomers are not sieved by the
gel, so they produce a diffuse “spot” instead of a band (A). (Capsomers can be separated into 2 bands by native polyacrylamide
gels, not shown). Migration in these agarose gels is dependent on both size and charge, so no “molecular weight standards” for
these gels are possible. The difference in migration due to charge changes is demonstrated by the behavior of the particles pro-
duced by mutant K169Y: both the K196Y proheads and K169Y heads migrate faster than their wild-type versions. The head form
of K169Y is called Head I because it doesn‘t have the covalent cross-links that stabilize HK97 Head II. Incorporation of the
(inactive) HK97 protease into proheads does not change the mobility because it is inside and its charge is not exposed.
ble monomer, but it does form a variety of higher order assemblies that can be visualized as bands in gels. Agarose gels are a
convenient way of discovering which assemblies are made by different mutants in vivo, or are produced by different treatments in
vitro. In agarose gels, fully assembled Prohead I (PI) and Prohead II (PII) make distinct bands that run at nearly the same position
(PII is slightly faster). Head II (HII), the expanded and mature HK97 capsid, runs slower than proheads in agarose gels, but so do
capsomers, which are the mixture of hexamers and pentamers made by dissociating Prohead I. Capsomers are not sieved by the
gel, so they produce a diffuse “spot” instead of a band (A). (Capsomers can be separated into 2 bands by native polyacrylamide
gels, not shown). Migration in these agarose gels is dependent on both size and charge, so no “molecular weight standards” for
these gels are possible. The difference in migration due to charge changes is demonstrated by the behavior of the particles pro-
duced by mutant K169Y: both the K196Y proheads and K169Y heads migrate faster than their wild-type versions. The head form
of K169Y is called Head I because it doesn‘t have the covalent cross-links that stabilize HK97 Head II. Incorporation of the
(inactive) HK97 protease into proheads does not change the mobility because it is inside and its charge is not exposed.
HK97 capsid proteins in SDS polyacrylamide gels. (C, D) HK97 gp5 is the 42 kDa major capsid protein, gp5* is the 31 kDa
cleaved product, and gp4 is the 25 kDa maturation protease (gp3 is the portal protein, but gp3 is not present in any of the samples
shown). SDS gels provide the means to distinguish between Prohead I and Prohead II: Prohead I has only 42 kDa gp5 and
Prohead II contains only 31 kDa gp5*. An inactive version of the protease, which has a mutation (H65A) in a catalytic residue, is
incorporated into proheads, but is unable to carry out the cleavage reaction. In Head II, the smallest forms of the major capsid pro-
tein are crosslinked forms with five or six monomers, the rest of the protein is in very large complexes that generally do not enter
the gel. Incubation in SDS sample buffer (before heating) fortuitously induces proheads to expand and cross-link (D), but this is
prevented by TCA precipitating the samples before denaturing in SDS (C). The fortuitous cross-linking induced by SDS sample
buffer creates ladders of bands that start with monomers and dimers and increase in size (D). Crosslinking cannot occur in free
capsomers and so the ability to induce such crosslinked ladders provides an indirect assay for assembly.
cleaved product, and gp4 is the 25 kDa maturation protease (gp3 is the portal protein, but gp3 is not present in any of the samples
shown). SDS gels provide the means to distinguish between Prohead I and Prohead II: Prohead I has only 42 kDa gp5 and
Prohead II contains only 31 kDa gp5*. An inactive version of the protease, which has a mutation (H65A) in a catalytic residue, is
incorporated into proheads, but is unable to carry out the cleavage reaction. In Head II, the smallest forms of the major capsid pro-
tein are crosslinked forms with five or six monomers, the rest of the protein is in very large complexes that generally do not enter
the gel. Incubation in SDS sample buffer (before heating) fortuitously induces proheads to expand and cross-link (D), but this is
prevented by TCA precipitating the samples before denaturing in SDS (C). The fortuitous cross-linking induced by SDS sample
buffer creates ladders of bands that start with monomers and dimers and increase in size (D). Crosslinking cannot occur in free
capsomers and so the ability to induce such crosslinked ladders provides an indirect assay for assembly.