Understanding and applications of Ser/Gly linkers in protein engineering

Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn2+

“…Indeed, for the 20–30 Å distance between the linker ends in the Zn 2+ -bound state of the sensor, higher effective concentrations are predicted for flexible linkers compared to polyproline linkers. 32 , 33 Furthermore, a decrease in effective linker length is expected upon increasing the length of the stiff polyproline linker, which is in line with the small but consistent increase in K D observed experimentally when increasing the length of the proline linker. Importantly, the attenuation of the Zn 2+ affinity from 160 pM in the parental BLZinCh-1 to the 0.5–1 nM affinities for the BLZinCh-Pro sensors makes the latter better suited for measuring intracellular cytosolic Zn 2+ and free Zn 2+ in serum.…”

“…Although the average distance between the fluorescent domains is larger in the Zn 2+ -depleted state than in the Zn 2+ -bound state, flexible linkers form relatively compact ensembles, giving rise to a substantial amount of energy transfer also in the Zn 2+ -depleted state. 32 Due to the high energy transfer efficiency in the Zn 2+ -bound state, this was not a problem when measuring FRET (ratiometric change of ∼400%), but it severely affected the BRET response. 23 Therefore, to increase the distance between the fluorescent proteins in the Zn 2+ -depleted state, we developed the BLZinCh-Pro series of bioluminescent Zn 2+ sensors in which the flexible (GGS) 18 linker was replaced by rigid polyproline linkers of different lengths.…”

Paving the way towards continuous biosensing by implementing affinity-based nanoswitches on state-dependent readout platforms

“…Notably, the binding epitopes were connected by a glycine-serine linker, which are commonly applied flexible spacers, and are therefore desirable to develop a protein clamp that switches between a flexible and rigid state upon target binding. 34 The abovementioned strategy was also applied by Adamson et al by connecting two Affimer proteins 35 that bind to human C-reactive protein (a protein biomarker), Herceptin (a therapeutic antibody) or the cow pea mosaic virus (a plant virus) respectively, thereby further demonstrating the generalizability of this approach to develop protein switches for multivalent targets. 21 Moreover, protein clamps have also been developed for various peptides, by sandwiching them between two distinct peptide binding interfaces that were selected through directed interface evolution.…”

“…22,64 Furthermore, for both protein and NA clamps, it was shown that by altering the length or stiffness of the linkers that attach both binders, the affinity of the switch can be tuned. 34,39,142,143 More specifically, the length and stiffness of the linkers will affect both the magnitude of the conformational change as well as the local effective concentration of the respective binders, which both affect the final signal that is generated for a certain target concentration. 34,39 This strategy was applied to enhance the affinity of protein and NA clamps for the detection of Ca( ii ), 144 K( i ), 145 Zn( ii ) 142 and immunoglobin heavy mu chain, 143 by screening different lengths and compositions of glycine-serine and PEG-linkers respectively.…”

“…34,39,142,143 More specifically, the length and stiffness of the linkers will affect both the magnitude of the conformational change as well as the local effective concentration of the respective binders, which both affect the final signal that is generated for a certain target concentration. 34,39 This strategy was applied to enhance the affinity of protein and NA clamps for the detection of Ca( ii ), 144 K( i ), 145 Zn( ii ) 142 and immunoglobin heavy mu chain, 143 by screening different lengths and compositions of glycine-serine and PEG-linkers respectively. 142–145 In this manner, the affinity could be enhanced from the pM to fM, 142 low to mid nM, 144 low to mid mM and low to mid μM 143 range for the Zn( ii ), 142 Ca( ii ), 144 K( i ) 145 and immunoglobin heavy mu chain 143 binding switches respectively.…”