Partially Folded Bovine Pancreatic Trypsin Inhibitor Analogues Attain Fully Native Structures when Co-Crystallized with S195A Rat Trypsin Irina V. Getun 1 , C. Kent Brown 2 , Judit Tulla-Puche 1 , Douglas Ohlendorf 2 , Clare Woodward 3 and George Barany 1 ⁎ 1 Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA 3 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, MN 55108, USA Received 23 July 2007; received in revised form 28 October 2007; accepted 30 October 2007 Available online 6 November 2007 Crystal structures, at 1.7 Å resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14–38] Abu retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric α-amino-n-butyric acid residues. The analogue K26P,A27D[14–38] Abu contains two further replacements, by statistically favored residues, in the type I β-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable. © 2007 Published by Elsevier Ltd. Edited by R. Huber Keywords: BPTI; [14–38] Abu ; partially folded protein; ligand-induced folding; crystal structure Introduction Partially folded proteins are likely to resemble transient metastable species formed during protein folding, 1–5 as well as natively unfolded proteins 6–15 and some disease-associated proteins. 16–25 Our laboratories have published extensively on ways to model partially folded (and also denatured) states derived from the protein bovine pancreatic trypsin inhibitor (BPTI), based on the replacement of cystine cross-links by pairwise α-amino-n-butyric acid (Abu) isosteres. 26,27 The appropriate chemi- cally synthesized full-length analogues allow us to access, without use of chemical denaturants, me- tals, or extremes of pH or temperature, species that are normally refractory to equilibrium char- acterization because their lifetimes are short com- pared to the time scale of conventional high- resolution structural techniques. Even so, the structures observed are heterologous and better *Corresponding author. E-mail address: barany@umn.edu. Present addresses: I. V. Getun, Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA; C. K. Brown, Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA; J. Tulla-Puche, Barcelona Biomedical Research Institute, Barcelona Science Park, 08028, Barcelona, Spain. Abbreviations used: Abu, α-amino-n-butyric acid; ANS, 8-anilino-1-naphthalenesulfonic acid; BPTI, bovine pancreatic trypsin inhibitor; CD, circular dichroism; HPLC, high-performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time of flight mass spectrometry; NMR, nuclear magnetic resonance; rmsd, root-mean-square deviation. doi:10.1016/j.jmb.2007.10.084 J. Mol. Biol. (2008) 375, 812–823 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Published by Elsevier Ltd.