Abstract
We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions. Published by Wiley-Blackwell.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1145-1154 |
| Number of pages | 10 |
| Journal | Protein Science |
| Volume | 20 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 2011 |
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Keywords
- Dissociation kinetics
- In vitro compartmentalization
- Loop
- Streptavidin
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding. / Magalhães, Maria L B; Czekster, Clarissa Melo; Guan, Rong; Malashkevich, Vladimir N.; Almo, Steven C.; Levy, Matthew.
In: Protein Science, Vol. 20, No. 7, 07.2011, p. 1145-1154.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding
AU - Magalhães, Maria L B
AU - Czekster, Clarissa Melo
AU - Guan, Rong
AU - Malashkevich, Vladimir N.
AU - Almo, Steven C.
AU - Levy, Matthew
PY - 2011/7
Y1 - 2011/7
N2 - We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions. Published by Wiley-Blackwell.
AB - We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions. Published by Wiley-Blackwell.
KW - Dissociation kinetics
KW - In vitro compartmentalization
KW - Loop
KW - Streptavidin
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UR - http://www.scopus.com/inward/citedby.url?scp=79959372166&partnerID=8YFLogxK
U2 - 10.1002/pro.642
DO - 10.1002/pro.642
M3 - Article
C2 - 21520321
AN - SCOPUS:79959372166
VL - 20
SP - 1145
EP - 1154
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 7
ER -