1. The Trp cage motif as a scaffold for the display of a randomized peptide library on bacteriophage T7
Richard E Herman, Douglas Badders, Mark Fuller, Ekaterina G Makienko, Michael E Houston Jr, Steven C Quay, Paul H Johnson J Biol Chem. 2007 Mar 30;282(13):9813-9824. doi: 10.1074/jbc.M610722200. Epub 2007 Jan 30.
Phage libraries displaying linear or disulfide-constrained peptides often yield weak binders, upon screening against a target, and must be optimized to improve affinity. The disadvantages of libraries based on larger complex proteins, such as single chain antibodies, have stimulated interest in the development of smaller nonimmunoglobulin protein scaffolds. A promising candidate is the Trp cage motif, a 20-residue C-terminal sequence of exendin-4. Amino acid substitution within the Trp cage resulted in a 20-mer peptide recognized as an ultrafast cooperative folding miniprotein, with ideal characteristics for the discovery of small structured nonimmunoglobulin motifs having a stable tertiary structure. Although we were unable to display the Trp cage on M13 phage, successful display was achieved using the lytic T7 phage. Interestingly, mutations were observed at a frequency dependent on display valency. A Trp cage library designed with randomized amino acids at seven solvent-exposed positions was developed from 1.6 x 10(9) primary clones in T7Select10-3b. DNA sequencing of 109 library clones revealed 38% mutants and 16% truncations by TAG codons at randomized positions. Amino acid frequencies were largely within expected bounds and DIVAA analysis revealed that the library had an average diversity of 0.67. Utility of the library was demonstrated by identification of HPQ containing Trp cage miniproteins, which bound streptavidin, and AAADPYAQWLQSMGPHSGRPPPR, which bound to human bronchial epithelial cells. A high complexity library based on the Trp cage miniprotein has demonstrated potential for identifying novel cell and protein binding peptides that could be used for the delivery of therapeutic molecules or as target-specific therapeutic agents.
2. The Trp-cage: optimizing the stability of a globular miniprotein
Bipasha Barua, Jasper C Lin, Victoria D Williams, Phillip Kummler, Jonathan W Neidigh, Niels H Andersen Protein Eng Des Sel. 2008 Mar;21(3):171-85. doi: 10.1093/protein/gzm082. Epub 2008 Jan 18.
The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).
3. Reversing the typical pH stability profile of the Trp-cage
Katherine A Graham, Aimee Byrne, Ruth Son, Niels H Andersen Biopolymers. 2019 Mar;110(3):e23260. doi: 10.1002/bip.23260. Epub 2019 Feb 19.
The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.