Engineering surface epitopes to improve protein crystallization
The invention provides for methods and systems for engineering target proteins, based on protein sequence characteristics that influence the likelihood of obtaining a crystal suitable for X-ray structure solution, to improve protein crystallization, as well as related material.
1. A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties, comprising:
a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
iii. using a computer program to predict the secondary structure of the target protein based on its sequence; and
iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein; and
c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces; and
d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence; and
e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system.
2. The method of claim 1 in which the sub-epitope library comprises the sequences in tables 1-38.
3. The method of claim 1 , in which the candidate sub-epitopes for substitution at the candidate sites is selected from tables 8 or 12.
4. A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties comprising:
a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
iii. using a computer program to predict the secondary structure of the target protein based on its sequence; and
iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein; and
c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces; and
d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence; and
e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system, and the expressed protein is crystallized and its structure is determined with high-resolution X-ray crystallography.
5. A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties, comprising
a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
iii. using a computer program to predict the secondary structure of the target protein based on its sequence; and
iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein; and
c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces; and
d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence; and
e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system to provide a mutant protein with the mutant protein sequence and is crystallized and its structure is determined with high-resolution X-ray crystallography.
6. The method of claim 5 , in which the sub-epitope library comprises the sequences in tables 1-38.
7. The method of claim 5 , in which the candidate sub-epitopes for substitution at the candidate sites is selected from tables 8 or 12.