Structural & Functional Characterization
of Engineered Avidin Proteins
Acta Universitatis Tamperensis,
No. 1529


By Juha Matta
July 2010
Tampere University Press
Distributed By Coronet Books
ISBN: 9789514481031
160 pages
$82.50 Paper Original

Avidin was first found in chicken, but is now known to be present in the avian egg-whites or egg-jellies. Avidin is thought to work as a defensive agent in the egg preventing the growth of unwanted organisms by creating a biotin drain. It binds its natural ligand, biotin, with very tight affinity. The determinants of this tight binding have been solved with the 3D structure of the avidin-biotin complex. It revealed that there are several hydrogen bonds between the avidin and biotin molecules and that the shape of the binding pocket is well optimized for biotin. Due to their tight affinity to biotin and good stability, avidin and its bacterial analogue streptavidin, have been used in a wide range of applications in biosciences. Collectively, applications based on avidin or streptavidin are referred to as strept(avidin)-biotin technology.

The aim of this study was to develop engineered forms of avidin and to characterise their properties. Avidin related proteins 1-7 (AVRs) belong to the avidin protein family. Their properties differ from each other and interestingly AVR4/5 was previously found to be more thermostable than avidin. To study the structural reasons behind the high thermostability of AVR4/5, we used genetic engineering to transfer parts of the sequence of AVR4/5 to avidin. As a result, we produced the chimeric avidin Ile117Tyr mutant (ChiAVD(I117Y)), the most thermostable avidin so far. Furthermore, we wanted to see if the high thermal stability of chimeric avidin correlated with stability in harsh organic solvents. Chimeric avidin was found equal or superior in stability when compared to avidin or streptavidin in most of the conditions studied. Therefore, chimeric avidin may prove useful in applications where high stability is required.

The loop region between the ?-strands 3 and 4 (L3,4) of avidin is important for ligand binding. This region was removed by circular permutation and the properties of the resulting deletion mutant were then compared to those of wild type avidin. The deletion of L3,4 combined with the point mutation Asp118Met (cp4?3(N118M)) reduced biotin binding affinity and increased affinity to 4?-hydroxyazobenzene-2-carboxylic acid (HABA). Based on isothermal titration calorimetry (ITC) analysis the relative HABA/biotin binding affinity of cp4?3(N118M) increased over a billion fold (109) when compared to that of wt avidin.

The third part of the study aimed at characterising the structure and properties of a novel chicken biotin-binding protein A (BBP-A). The structure of the protein in complex with the ligand revealed that the ligand-binding site was occupied by d-biotin d-sulfoxide (BSO). ITC analysis confirmed that BBP-A binds both BTN and BSO with comparable affinities, a property unique among avidins. The structure of the protein resembles that of avidin, but its thermostability is lower.

In summary, this multidisciplinary study clarified factors affecting the ligand-binding properties of avidin and novel avidin-like proteins, and therefore, increased the knowledge of the structure-function relationship of avidins. Furthermore, this work may offer new tools for avidin-biotin technology.

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