The sli
ding cla
mps of bacteriophage T4 (gp45),
Escherichia coli (
![](/i<font color=)
mages/gifchars/beta2.gif" BORDER=0 ALIGN="
mi
ddle"> cla
mp), an
d yeast (PCNA)are require
d for processive DNA synthesis by their cognate DNA poly
merases. The X-ray crystal structuresof all three of these cla
mps have been shown to be close
d, circular co
mplexes. This paper reportsinvestigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation,fluorescence, an
d hy
dro
dyna
mic
mo
deling. Mutants of gp45 with inter- an
d intrasubunit
disulfi
de bon
dswere create
d to alter the solution structure of gp45, with a
dditional
mutagenesis use
d to investigate thei
mportance of the proline-rich loop region foun
d between the two
do
mains of each gp45
mono
mer. Thewil
d-type gp45 tri
mer asse
mbles fro
m mono
mers cooperatively with a
dissociation constant of 0.21
![](/i<font color=)
mages/entities/
mgr.gif">M
2an
d values between 0.088 an
d 0.32
![](/i<font color=)
mages/entities/
mgr.gif">M
2 for the
mutants. Velocity ultracentrifugation experi
ments showe
dthat wil
d-type gp45 possesses a se
di
mentation coefficient strongly
depen
dent on concentration, typical ofasy
mmetric or elongate
d molecules, that when extrapolate
d to zero concentration yiel
ds a se
di
mentationcoefficient of 4.0 S. The loop an
d the
disulfi
de
mutants exhibite
d se
di
mentation coefficients with littleconcentration
depen
dence, typical of sy
mmetric or spherical
molecules, that when extrapolate
d to zeroconcentration yiel
de
d se
di
mentation coefficients of 4.4-4.8 S. The lower se
di
mentation coefficient inthe for
mer case is consistent with wil
d-type gp45 being
more asy
mmetric or elongate
d than the
mutantfor
ms. Fluorescence-resonance energy-transfer experi
ments were use
d to
measure the
distance betweentwo a
mino aci
ds (W91 an
d V162C-cou
marin) on opposite si
des of the gp45 subunit interface. For anintrasubunit
disulfi
de
mutant, the
distance between these two a
mino aci
ds was
deter
mine
d to be 19 Å (14Å in the X-ray crystal structure), consistent with a close
d co
mplex. For the
mutants without intrasubunit
disulfi
des, the efficiency of fluorescence-resonance energy transfer was in accor
d with a
mo
del of gp45being an open co
mplex co
mpose
d of two close
d subunit interfaces an
d a thir
d open interface separate
dby a
distance of 35-38 Å. The collective
data supple
mente
d with hy
dro
dyna
mic
mo
deling were consistentwith gp45 subunit separation achieve
d within the plane of the gp45 ring.