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Design and engineering of alpha-helical bundle proteins: modified structures and novel functions

Objective

The goal is to understand the structural principles of small globular proteins as a basis for engineering novel functions and to apply this understanding to the production of specific prototypes of engineered proteins. Functions to be engineered into the helical bundle proteins include metal binding sites, enzyme inhibitors, peptide hormone analogues and epitope carriers.
The collaborative group has concentrated on developing basic protein engineering techniques. The research vehicle is a particularly simple type of protein structure that occurs many times in nature as a basic protein building block and is made of four cylindrical substructures. using a variety of techniques ranging from genetic engineering to biochemical and physical methods and design by computer software several new engineering principles for protein molecules have been explored. New functional pieces were introduced into the natural protein frameworks. One helps the proteins form regular crystalline arrays. Another is a medically important activator of the immune system which can be repackaged in possibly useful ways in the attempt to develop new vaccines. A third design is an unexpected rearrangement of the threading of the protein chain which nonetheless spontaneously forms the correct three-dimensional shape. A fourth design demonstrates how the amplification power of natural selection can be put at the disposal of the protein engineer: a genetic switch element was coupled to a protein fragment such that only correctly assembled protein fragments will allow cells to grow, amplifying and selecting out the best molecules.

The immediate objections were to understand by a combination of theory and experiment the structural principles of the 4-alpha-helix bundle proteins rop and ferritin as a basis for engineering novel functions. Variations to be engineered include core and loop changes, transfer of a ferroxidase site and introduction of an immunogenic epitope. Where possible, 3-dimensional structures were to be determined by crystallography.

Research accomplishments to date xx include:
the first successful design of a metal binding site that helps proteins (ferritin) crystallize;
the first successful topological reengineering of a protein (Rop) that goes beyond cyclic shift of chain ends;
engineering of a highly immunogenic protein by genetic grafting of a peptide, (a protein fragment) from the medically important cytokine interleukin 1 beta onto a protein (ferritin) that assembles into higher order structures;
engineering of a hybrid between Rop protein and the deoxyribonucleic acid (DNA) binding domain of a genetic switch element (the lambda repressor), such that ROP acts like the activating principle (dimerization domain) of the genetic switch. Cells that have a well folded Rop grow better than those which do not.
In order to develop the full potential of protein engineering, fundamental aspects of protein 3-D structure must be Well understood. The 4-alpha-helix bundle is a simple, recurrent structural motif that can incorporate various functions and can occur in different assemblies. The structural simplicity and functional diversity of this protein motif makes it an excellent candidate for protein engineering exercises.
Our immediate goals are to understand, by a combination of theory and experiment, the structural principles of these proteins as a basis for engineering novel functions; and, to apply this understanding to the production of specific prototypes of engineered functional proteins. Functions to be engineered include metal binding sites, enzyme inhibitors, peptide hormone analogues and epitope carriers.
We have assembled an international group of laboratories With complementary expertise in: gene synthesis and mutagenesis, construction of expression vectors, protein purification, functional and immunoassays, X-ray crystallography, NMR, light spectroscopy, energetics of Protein folding, micro-calorimetry, molecular modelling and dynamics, database searches and protein structure theory.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Address
Meyerhofstrasse 1
Heidelberg
Germany

Participants (6)

FOUNDATION OF RESEARCH AND TECHNOLOGY - HELLAS
Greece
Address
Vassilika Vouton
71110 Iraklion
Gesellschaft für Biotechnologische Forschung mbH
Germany
Address
Mascheroder Weg 1
38124 Braunschweig
THE UNIVERSITY OF MILANO
Italy
Address
Lita, Via Fratelli Cervi 93
20090 Segrate
UNIVERSITY OF SHEFFIELD
United Kingdom
Address
Western Bank
S10 2TN Sheffield
UNIVERSITÀ DEGLI STUDI DI ROMA TOR VERGATA
Italy
Address
Via Della Ricerca Scientifica, 1 Edificio Sogene
00133 Roma
WESTFAELISCHE WILHELMS-UNIVERSITAET MUENSTER
Germany
Address
Rontgenstrasse 9-13
48149 Muenster