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Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization
Author(s) -
Ross Philip D,
Cheng Naiqian,
Conway James F,
Firek Brian A,
Hendrix Roger W,
Duda Robert L,
Steven Alasdair C
Publication year - 2005
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7600613
Subject(s) - capsid , chemistry , biophysics , protein subunit , crystallography , differential scanning calorimetry , thermal stability , biochemistry , biology , thermodynamics , gene , physics , organic chemistry
In HK97 capsid maturation, structural change (‘expansion’) is accompanied by formation of covalent crosslinks, connecting residue K169 in the ‘E‐loop’ of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead‐II passes through three expansion intermediate (EI) states en route to the end state, Head‐II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild‐type and K169Y capsids. After expansion, the denaturation temperature ( T p ) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (Δ T p , +11°C). EI‐II is the earliest capsid to form crosslinks. Cryo‐electron microscopy shows that for both wild‐type and K169Y EI‐II, most E‐loops are in the ‘up’ position, 30 Å from the nearest N356: thus, crosslinking in EI‐II represents capture of mobile E‐loops in ‘down’ positions. At pH 4, most K169Y capsids remain as EI‐II, whereas wild‐type capsids proceed to EI‐III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.