TY - JOUR
T1 - Importance of Contact Persistence in Denatured State Loop Formation
T2 - Kinetic Insights into Sequence Effects on Nucleation Early in Folding
AU - Tzul, Franco O.
AU - Bowler, Bruce E.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant GM074750 (to B.E.B.).
PY - 2009/7/3
Y1 - 2009/7/3
N2 - Protein folding is dependent on the formation and persistence of simple loops early in folding. Ease of loop formation and persistence is believed to be dependent on the steric interactions of the residues involved in loop formation. We have previously investigated this factor in the denatured state of iso-1-cytochrome c using a five-amino-acid insert in front of a unique histidine in the N-terminal region of the protein. Previously, we reported that the apparent pKa values of loop formation for the most flexible (all Gly) and least flexible (all Ala) insert were, within error, the same. We evaluate whether this observation is due to differences in the persistence of loop contacts or due to effects of local sequence sterics and main-chain hydration on the persistence length of the chain. We also test whether sequence order affects loop formation. Here, we report kinetic results coupled to further mutagenesis of the insert to discern between these possibilities. We find that the amino acid-glycine versus alanine-next to the loop forming histidine has a dominant effect on loop kinetics and equilibria. A glycine in this position speeds loop breakage relative to alanine, resulting in less stable loops. At high percentage of Gly in the insert, rates of loop formation and breakage exactly compensate, leading to a leveling out in loop stability. Loop formation rates also increase with glycine content, inconsistent with poly-Gly segments being more extended than previously suspected due to main-chain hydration or local sterics. Unlike loop breakage rates, loop formation rates are insensitive to local sequence. Together, these observations suggest that contact persistence plays a more important role in defining the "folding code" than rates of loop formation.
AB - Protein folding is dependent on the formation and persistence of simple loops early in folding. Ease of loop formation and persistence is believed to be dependent on the steric interactions of the residues involved in loop formation. We have previously investigated this factor in the denatured state of iso-1-cytochrome c using a five-amino-acid insert in front of a unique histidine in the N-terminal region of the protein. Previously, we reported that the apparent pKa values of loop formation for the most flexible (all Gly) and least flexible (all Ala) insert were, within error, the same. We evaluate whether this observation is due to differences in the persistence of loop contacts or due to effects of local sequence sterics and main-chain hydration on the persistence length of the chain. We also test whether sequence order affects loop formation. Here, we report kinetic results coupled to further mutagenesis of the insert to discern between these possibilities. We find that the amino acid-glycine versus alanine-next to the loop forming histidine has a dominant effect on loop kinetics and equilibria. A glycine in this position speeds loop breakage relative to alanine, resulting in less stable loops. At high percentage of Gly in the insert, rates of loop formation and breakage exactly compensate, leading to a leveling out in loop stability. Loop formation rates also increase with glycine content, inconsistent with poly-Gly segments being more extended than previously suspected due to main-chain hydration or local sterics. Unlike loop breakage rates, loop formation rates are insensitive to local sequence. Together, these observations suggest that contact persistence plays a more important role in defining the "folding code" than rates of loop formation.
KW - denatured states
KW - loop formation
KW - main-chain flexibility
KW - protein folding
KW - sequence composition
UR - http://www.scopus.com/inward/record.url?scp=67349120000&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2009.04.075
DO - 10.1016/j.jmb.2009.04.075
M3 - Article
C2 - 19426739
AN - SCOPUS:67349120000
SN - 0022-2836
VL - 390
SP - 124
EP - 134
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -