Conversion of Type I 4:6 to 3:5 -Turn Types in Human Acidic Fibroblast Growth Factor: Effects upon Structure, Stability, Folding, and Mitogenic Function Jihun Lee, 1 Vikash Kumar Dubey, 2 Thayumanasamy Somasundaram, 3 and Michael Blaber 2 * 1 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4300 2 Department of Biomedical Sciences, Florida State University, Tallahassee, Florida 32306-4300 3 Kasha Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4300 ABSTRACT Human acidic fibroblast growth factor (FGF-1) is a member of the -trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 con- tains 11 -turns, the majority being type I 3:5; how- ever, a type I 4:6 turn is also found at three symmetry- related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of dele- tion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional se- quence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local envi- ronment). Although the results suggest that a stable, foldable -trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function. Proteins 2006;62:686 – 697. © 2005 Wiley-Liss, Inc. Key words: -hairpin; protein engineering; protein stability; protein folding; FGF INTRODUCTION Protein architectures are built up from -helical, -strand, and turn secondary structure motifs. Among these secondary structures, -turns are the most common type of nonrepetitive structure recognized in proteins, and comprise about 25% of the residues. 1 Despite this abun- dance, less is understood regarding the contribution of turns to the structure, stability, and folding of proteins than for -helices and -sheets. One reason for this lack of knowledge is that -turns appear to be far more varied and complex than either -helices or -sheets. Richardson and coworkers 2 have characterized the -turn as having six distinct types (I, I, II, II, VIa, and VIb), as well as a miscellaneous category (IV), based on main-chain ,  angles. If a -turn connects two -strands that form an antiparallel -sheet, then it is known as a -hairpin turn. Thornton and coworkers 3 developed an “X:Y” shorthand for -hairpin structures whereby X identi- fies the number of residues required for the turn and Y identifies the hydrogen bonding pattern in the closure of the turn (X equals Y when the closure involves two backbone hydrogen bonds, whereas Y  X  2 if closure involves one hydrogen bond). Thornton noted that the number of residues in -hairpin turns typically comprises two to seven residues (X  2–7), and can be further classified according to different patterns of backbone hydro- gen bonding. This complexity makes -turns a fertile area for biophysical studies of protein stability and folding. Various reports have characterized the relationship between turn residues and -hairpin formation using small peptide systems. 4–8 Jimenez and coworkers 9 showed that different turn sequences can alter turn conformation, and consequently, the -sheet registration of -hairpins. Conversely, Searle and coworkers 10 reported that turn regions within a protein may be able to maintain their conformation despite variations in their primary struc- ture. Rotondi and Gierasch 8 have shown that two of the seven -hairpin turns in retinoic acid binding protein I form spontaneously in solution as short peptides, suggest- Abbreviations: FGF, fibroblast growth factor; ASU, asymmetric unit; FGFR, FGF receptor; ADA, N-(2-acetamido)iminodiacetic acid; DTT, dithiothreitol; PEG, polyethylene glycol; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid; GuHCl, guanidine hydrochlo- ride; DMEM, Dulbecco’s modified Eagle’s medium; NCS, newborn calf serum; TBS, Tris-buffered saline; RMSD, root-mean-square deviation; CPK, Corey, Pauling, Koltun. *The triple letter abbreviations for amino acids are used throughout the manuscript; however, for conciseness the single letter amino acid code is substituted when referring to certain mutations and for listing of amino sequence information. Deletion mutations are symbolized using Greek delta () followed by the residue type and position. Substitution mutations are referenced by providing the wild-type amino acid (triple or single-letter code), the amino acid position, and then the mutant amino acid. Grant sponsor: the National Science Foundation; Grant number: MCB 0314740. †Correspondence to: Michael Blaber, Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306-4300. E-mail: michael.blaber@med.fsu.edu Received 12 July 2005; Accepted 21 September 2005 Published online 14 December 2005 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.20808 PROTEINS: Structure, Function, and Bioinformatics 62:686 – 697 (2006) © 2005 WILEY-LISS, INC.