Nucleic Acids Research 2004 32(Web Server Issue):W562-W565; doi:10.1093/nar/gkh422
© 2004, the authors
Nucleic Acids Research, Vol. 32, Web Server issue © Oxford University Press 2004; all rights reserved
LINKER: a web server to generate peptide sequences with extended conformation
Fan Xue1,
Zhong Gu2 and
Jin-an Feng1,2,*
1 Department of Chemistry and 2 Center for Biotechnology, Temple University, 1901 N 13th Street, Philadelphia, PA 19122, USA
* To whom correspondence should be addressed at Department of Chemistry, Temple University, 1901 N 13th Street, Philadelphia, PA 19122, USA. Tel: +1 215 204 7128; Fax: +1 215 204 1532; E-mail: feng{at}astro.temple.edu
Received February 14, 2004; Revised and Accepted April 8, 2004
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ABSTRACT
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LINKER was developed as an online server to assist biomedical
researchers to design linker sequences for constructing functional
fusion proteins. The program automatically generates a set of
peptide sequences that are known to adopt extended conformations
as determined by X-ray crystallography and NMR. In addition
to the desired linker sequence length, the web interface provides
a number of optional input parameters so that the users may
enhance sequence selection based on the requirements of specific
applications. The output of LINKER includes a list of peptide
sequences with specified length and sequence characteristics.
A graphical subroutine was implemented to highlight the chemical
features of every linker sequence by hydrophobicity plots. LINKER
can be accessed at
http://astro.temple.edu/~feng/Servers/BioinformaticServers.htm.
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INTRODUCTION
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The gene fusion technique has become an increasingly useful
tool in biomedical research. Fusion proteins have been constructed
to increase cellular stability and biological activity of functional
proteins (
1). In structural biology, the construction of recombinant
fusion protein has often been used as a means to increase the
expression of soluble proteins and to facilitate protein purification
(
2). In biotechnology, fusion proteins have been constructed
to engineer bifunctional enzymes, and to select and produce
antibodies (
3–
5). Rationally designed P450 enzymes were
produced by gene fusion of the different domains of the cytochrome
P450 BM3 from
Bacillus megaterium (BMP) with flavodoxin and
the domains of human P450 2E1 (
6). These engineered fusion proteins
showed improved solubility and electrochemical properties with
catalytic activities of the P450 enzymes. Specialized functional
fusion proteins have been constructed to target specific genes
(
7,
8). It is expected that the gene fusion technology will continue
to have significant impact on a variety of fields of biomedical
research.
The construction of a fusion protein involves the linking of two macromolecules by a linker sequence. The selection of the linker sequence is of particular importance. Linker sequence composition could affect the folding stability of a fusion protein. It is often unfavorable to have a linker sequence with high propensity to adopt 
-helix or ß-strand structures, which could limit the flexibility of the protein and consequently its functional activity. Indeed, a more desirable linker is a sequence with preference to adopt extended conformation. In practice, most currently designed linker sequences have a high content of glycine residues that force the linker to adopt loop conformation. However, such monotonous linkers limit the functional activity of the fusion proteins. With the rapid advancing of biotechnology, it is envisioned that more sophisticated fusion proteins with diversified linker sequences will be created.
Polypeptide sequences specify their adopted secondary structures in proteins. Sequence analyses of proteins have revealed unique sequence patterns favorable to adopt -helix and loop conformations (9,10). The loop sequences are actually quite diversified in proteins (9). It was found that a variety of loop sequences exist to accommodate different chemical environments of protein structures. For example, the surface loops in proteins are more hydrophilic while the interior loops of the proteins are relatively neutral. Loops of different sizes also exhibit different sequence patterns. Taking advantage of the knowledge learned from analysis of protein structures, we developed a program called LINKER that automatically generates a set of peptide sequences that are known to adopt extended conformations as determined by X-ray crystallography and NMR. A loop library was derived from the Protein Data Bank (PDB) that contained both globular and membrane proteins. Loop sequences of various lengths were extracted as previously described (11). Loop lengths of less than four residues, as well as the redundant loop sequences, were also removed from the library. LINKER is a web-based server that allows the user to select sequences of their choice.
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LINKER SERVER
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The goal to the design of LINKER was to provide a simple user
interface for easy interaction. The workflow of the program
can be described as a series of sequence filters (
Figure 1)
(
11). The first filter, an input of the desired linker sequence
length, directs the program to search the loop library and fetch
linker sequences that match the input requirement. The second
filter removes linker sequences that may contain sequence patterns
sensitive to specified proteases. In constructing the fusion
gene, it is often necessary to process the ends of the fusing
genes and the linker gene sequence with restriction enzymes
before ligating them together genetically. Linker sequences
containing nucleotide sites sensitive to the same restriction
enzyme as that of ends processing enzymes would be unfavorable
choices. The third filter of LINKER allows the user to incorporate
this consideration in their experimental design. LINKER also
gives users the ability to select the chemical feature of the
linker sequences by specifying desired amino acid compositions.
The required restriction enzyme ends processing also limits
the selection of amino acids for both ends of linker sequences,
since restriction sites only encode corresponding amino acids.
Users may specify this limitation in the input page of LINKER.
Figure 2(a) shows the input page of LINKER. The user may specify
the length of desired linker sequence with either the number
of residues or the estimated length in angstroms. Proteolytic
sites for six most commonly used proteases were incorporated
in the LINKER. They include trypsin, chymotrypsin, thrombin,
plasmin, papain and factor Xa. To activate this filter, the
user may simply select the box next to the proteases. Alternatively,
the user may select linker sequences that contain desirable
protease sensitive sites. The input page also displays a complete
list of all the restriction sites that are incorporated in the
LINKER. The user may select the restriction enzymes used in
the fusion gene construction. The program will automatically
avoid selecting linker sequences that contain these restriction
sites in their respective coding genes. In the amino acid composition
filter, we implemented three selection categories in order to
help users to specify their selections. For example, in constructing
a membrane fusion protein, it is perhaps more favorable to include
linker sequences with hydrophobic residues. On the other hand,
in constructing a DNA-binding protein, it may be desirable to
exclude highly charged residues (Lys and Arg) in the linker
sequences since these residues could form salt bridges with
the phosphate backbone of the DNA, thus influencing the DNA-binding
property of the engineered fusion protein.
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Figure 2. An intuitive user interface of LINKER. (a) The input page of the LINKER with a layout of various input options; (b) an example of LINKER output with linker sequences of 45 residues.
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The output of LINKER is simple and easy to inspect [
Figure 2(b)].
It contains a list of sequences that are known to adopt extended
conformation. A PDB access code was placed at the beginning
of each sequence identifying the source of the linker sequence.
In this updated version of LINKER, we implemented a graphical
representation of the chemical features of each sequence by
hydrophobicity profiling. The hydrophobicity index for each
amino acid was obtained from the Eisenberg consensus hydrophobicity
scale (
12).
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AVAILABILITY
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LINKER was written in Fortran with CGI interface. It was compiled
on a LINUX-based workstation. The web server can be freely accessed
on the World Wide Web at
http://astro.temple.edu/~feng/Servers/BioinformaticServers.htm.
A brief description of the program and a detailed user guide
with examples are also available at the website.
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Notes
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The online version of this article has been published under
an open access model. Users are entitled to use, reproduce,
disseminate, or display the open access version of this article
provided that: the original authorship is properly and fully
attributed; the Journal and Oxford University Press are attributed
as the original place of publication with the correct citation
details given; if an article is subsequently reproduced or disseminated
not in its entirety but only in part or as a derivative work
this must be clearly indicated.
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