Controlling Quaternary Structure Assembly: Subunit Interface Engineering and Crystal Structure of Dual Chain Avidin

Hytönen, Vesa P.; Hörhä, Jarno; Airenne, Tomi T.; Niskanen, Einari A.; Helttunen, Kaisa; Johnson, Mark S.; Salminen, Tiina A.; Kulomaa, Markku S.; Nordlund, Henri R.

doi:10.1016/j.jmb.2006.04.044

Cited by 14 publications

(16 citation statements)

References 34 publications

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“…For example, tandem repeats of protein domains have been designed effectively by fusing the proximal N-and C-termini of neighboring molecules. This type of duplication event results in repetition of the same fold (Brucker, 2000;Hytönen et al, 2006;Nauli et al, 2007;Zhou et al 2008). It has also been shown that when the N-and C-termini of the same protein are proximal, these termini can be linked together and new termini can be introduced in a number of different possible locations (Luger et al, 1989;Hennecke et al, 1999;Graf & Schachman, 1996;Iwakura et al, 2000).…”

Section: Figurementioning

confidence: 99%

Protein design by fusion: implications for protein structure prediction and evolution

Skorupka

Han

Nam

et al. 2013

Acta Crystallogr D Biol Cryst

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Domain fusion is a useful tool in protein design. Here, the structure of a fusion of the heterodimeric flagella-assembly proteins FliS and FliC is reported. Although the ability of the fusion protein to maintain the structure of the heterodimer may be apparent, threading-based structural predictions do not properly fuse the heterodimer. Additional examples of naturally occurring heterodimers that are homologous to full-length proteins were identified. These examples highlight that the designed protein was engineered by the same tools as used in the natural evolution of proteins and that heterodimeric structures contain a wealth of information, currently unused, that can improve structural predictions.

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Section: Figurementioning

confidence: 99%

Protein design by fusion: implications for protein structure prediction and evolution

Skorupka

Han

Nam

et al. 2013

Acta Crystallogr D Biol Cryst

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“…However, the latter circularly permuted avidin, cpAvd6 → 5, showed slightly reduced binding affinity, which may be caused by the increased freedom of the biotin-binding residues close to the termini to move. Another potential explanation is the unique conformation of Phe72 in cpAvd6 → 5 compared with other avidin forms [74]. Dual chain avidin (dcAvd) was modified further to contain two distinct independent ligand-binding sites or domains within a single polypeptide chain.…”

Section: Topology Modificationsmentioning

confidence: 99%

Genetically engineered avidins and streptavidins

Laitinen

Hytönen

Nordlund

et al. 2006

Cell. Mol. Life Sci.

290

197

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Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, K(d) approximately 10(-14)-10(-16) M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology.

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“…Similarly to dcAvd(I117C 5→4, V115H 6→5 ), dcAvd-Cys was a monomer at room temperature (RT, 23±1°C) in the absence of biotin. The presence of biotin stabilized dcAvd-Cys, although it lost its pseudotetrameric structure at 60°C, which is about 20°C lower than the thermal transition temperature determined for dcAvd(I117C 5→4, V115H 6→5 ) [20].…”

Section: Resultsmentioning

confidence: 59%

“…Therefore, it seems that maleimide was able to inhibit the binding of biotin to the cysteine-modified binding site in the cpAvd6→5 domain of the dcAvd-Cys. As a consequence, the remaining biotin-binding activity of dcAvd-Cys would depend mainly on the cpAvd5→4 domain, which closely resembles that of wt avidin [17], [20]. The rapid burst (∼10%) in the recovery of fluorescence just after the addition of free biotin was most probably mainly associated with the dissociation of fluorescent biotin from the cpAvd6→5 domain with the S16C modified binding site.…”

Section: Resultsmentioning

confidence: 96%

“…The subunits can independently be modified, making it possible to create avidins with altered binding sites, for instance for low-affinity and high-affinity binding of a ligand within a single protein molecule [7]. The use of a disulphide bridge (I117C 5→4 , numbering according to wt avidin) combined with a critical mutation (V115H 6→5 ) in the interface between subunits [19] made it possible to develop dcAvds with defined quaternary structures [20]. DcAvd provides a scaffold that can be modified not only to alternate affinity for a ligand, but also to change specificity – in other words to favour another ligand molecule instead of biotin.…”

Section: Introductionmentioning

confidence: 99%

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Bifunctional Avidin with Covalently Modifiable Ligand Binding Site

et al. 2011

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The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

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Controlling Quaternary Structure Assembly: Subunit Interface Engineering and Crystal Structure of Dual Chain Avidin

Cited by 14 publications

References 34 publications

Protein design by fusion: implications for protein structure prediction and evolution

Protein design by fusion: implications for protein structure prediction and evolution

Genetically engineered avidins and streptavidins

Bifunctional Avidin with Covalently Modifiable Ligand Binding Site

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