Circular permutation as a tool to reduce surface entropy triggers crystallization of the signal recognition particle receptor beta subunit.
The production of diffraction-quality crystals remains a difficult obstacle on the road to high-resolution structural characterization of proteins. This is primarily a result of the empirical nature of the process. Although crystallization is not predictable, factors inhibiting it are well established. First, crystal formation is always entropically unfavorable. Reducing the entropic cost of crystallizing a given protein is thus desirable. It is common practice to map boundaries and remove unstructured regions surrounding the folded protein domain. However, a problem arises when flexible regions are not at the boundaries but within a domain. Such regions cannot be deleted without adding new restraints to the domain. We encountered this problem during an attempt to crystallize the beta subunit of the eukaryotic signal recognition particle (SRbeta), bearing a long and flexible internal loop. Native SRbeta did not crystallize. However, after circularly permuting the protein by connecting the spatially close N and C termini with a short heptapeptide linker GGGSGGG and removing 26 highly flexible loop residues within the domain, we obtained diffraction-quality crystals. This protein-engineering method is simple and should be applicable to other proteins, especially because N and C termini of protein domains are often close in space. The success of this method profits from prior knowledge of the domain fold, which is becoming increasingly common in today's postgenomic era.
Schwartz
Thomas U
TU
Howard Hughes Medical Institute, Laboratory of Cell Biology, The Rockefeller University, New York, New York 10021, USA. tus@mit.edu
Walczak
Rudolf
R
Blobel
Günter
G
D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D019277:Entropy; D017354:Point Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics*; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000235:genetics*
D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; C036318:signal peptide receptor