Affilin–Novel Binding Molecules Based on Human
γ-B-Crystallin, an All β-Sheet Protein
Hilmar Ebersbach
1
⁎
, Erik Fiedler
1
, Tanja Scheuermann
1
Markus Fiedler
1
, Milton T. Stubbs
2,3
, Carola Reimann
1
Gabriele Proetzel
1
, Rainer Rudolph
2,3
and Ulrike Fiedler
1
1
Scil Proteins GmbH,
Heinrich Damerow Str. 1,
06120 Halle (Saale), Germany
2
Institut für Biochemie
und Biotechnologie,
Martin-Luther-Universität
Halle-Wittenberg,
Kurt-Mothes-Str. 3,
D-06120 Halle (Saale),
Germany
3
Mitteldeutsches Zentrum für
Struktur und Dynamik der
Proteine, Martin-Luther-
Universität Halle-Wittenberg,
Kurt-Mothes-Str. 3, D-06120
Halle (Saale), Germany
The concept of novel binding proteins as an alternative to antibodies has
undergone rapid development and is now ready for practical use in a wide
range of applications. Alternative binding proteins, based on suitable
scaffolds with desirable properties, are selected from combinatorial libraries
in vitro. Here, we describe an approach using a β-sheet of human γ-B-
crystallin to generate a universal binding site through randomization of
eight solvent-exposed amino acid residues selected according to structural
and sequence analyses. Specific variants, so-called Affilin, have been
isolated from a phage display library against a variety of targets that differ
considerably in size and structure. The isolated Affilin variants can be
produced in Escherichia coli as soluble proteins and have a high level of
thermodynamic stability. The crystal structures of the human wild-type γ-B-
crystallin and a selected Affilin variant have been determined to 1.7 Å and
2.0 Å resolution, respectively. Comparison of the two molecules indicates
that the human γ-B-crystallin tolerates amino acid exchanges with no major
structural change. We conclude that the intrinsically stable and easily
expressed γ-B-crystallin provides a suitable framework for the generation of
novel binding molecules.
© 2007 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: γ-B-crystallin; scaffold; de novo binding; intrinsic stability;
structure
Introduction
Molecular recognition is essential to regulating
biological processes. Key regulators are proteins,
which are able to enter into an immense number of
molecular interactions with small molecules, nucleic
acids, polysaccharides, lipids and of course with
each other through virtue of their chemical, struc-
tural and dynamic versatility.
The best characterized binding molecules in
nature are antibodies. A practically rigid and highly
conserved framework supports six hypervariable
loops, the complementarity-determining regions
(CDRs), whose high degree of sequence variability
and structural diversity are responsible for specific
binding of the antigen. Individual sequences of the
CDR loops are selected by affinity maturation to
match the corresponding features of the antigen
epitope, acting as a universal binding site of the
antibody. The broad binding repertoire of the
antibodies is increased further by the conforma-
tional diversity of the flexible CDRs, with one
sequence capable of adopting multiple structures.
1
Conformational changes may occur upon binding to
the antigen, and can consist of simple side-chain
movements, concerted movements of individual
Present addresses: H. Ebersbach, University of Zürich,
Department of Biochemistry, Winterthurerstrasse 190,
CH-8057 Zürich, Switzerland; G. Proetzel, The Jackson
Laboratory, 610 Main St., Bar Harbor, ME 04609, USA.
Abbreviations used: CDR, complementarity-
determining region; GdmHCl, guanidinium
hydrochloride; IMAC, immobilized metal-affinity
chromatography; MIA, melanoma inhibitory activity;
NGF, nerve growth factor; proNGF, pro-form of NGF;
SPC-x-xx, named Affilin molecule; SPR, surface plasmon
resonance; TMB, 3, 3′,5′,5′-tetramethylbenzidine.
E-mail address of the corresponding author:
hebersbach@bioc.unizh.ch
doi:10.1016/j.jmb.2007.06.045 J. Mol. Biol. (2007) 372, 172–185
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.