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The tryptophan-cage (Trp-cage) is a stability folded mini protein

The tryptophan-cage or "Trp-cage" is thought to be the smallest protein-like folding motif. The Trp-cage mini proteins are small, folded peptides with 18 to 20 residues in length. A 20 residue Trp-cage mini protein adopts a stable folded structure with well-defined secondary structure elements containing a hydrophobic core arranged around a single central Trp residue. The peptide can fold spontaneously into a stable 3D structure within ~4 µs. Qui et al. utilized laser temperature jump spectroscopy to measure the folding speed of the peptide. The observed fast-folding speed exceeded contact-order predictions and approached diffusional “speed limits” for protein folding.

The amino acid sequence of a protein determines the protein's final conformation or folding. A protein fold usually refers to a protein's native three-dimensional structure. The proper function of a protein in its native environment requires a correctly folded protein.

Since folding mechanisms of proteins are still not well understood, the Trp-cage mini protein is a model peptide for protein folding studies using bio-physical and bioinformatical methods such as laser temperature jump spectroscopy, differential scanning calorimetry, circular dichroism spectroscopy, and molecular dynamics simulations. 


Structural models of Trp-cage mini proteins.

 Structure and Sequence

 Notes

[1] TC5b mini protein: 

Neidigh et al. truncated and mutated a poorly folded 39-residue peptide to produce a 20-residue constructs that are >95% folded in water at physiological pH (power of hydrogen).

The peptides contained an optimized novel fold now known as the 'Trp-cage' motif, also known as stable mini proteins. The folding of these peptides is cooperative and hydrophobically driven by the encapsulation of a tryptophan side chain in a sheath of proline rings. Trp-cage mini proteins are now well studied and Neidigh et al. suggested that the Pro:Trp interactions present an effective strategy for the a priori design of self-folding peptides.

 >pdb|1L2Y|A Chain A, TC5b
  NLYIQWLKDGGPSSGRPPPS

Neidigh JW, Fesinmeyer RM, Andersen NH. Designing a 20-residue protein. Nat Struct Biol. 2002 Jun;9(6):425-30. doi: 10.1038/nsb798. PMID: 11979279.

[2] E6-binding peptides

Liu et al. designed monomeric E6-binding peptides that are stable in aqueous solution. The research group used a protein grafting approach to incorporate the E6-binding motif of E6-associated protein, E6AP, LQELLGE, into exposed helices of stably folded peptide scaffolds. Furthermore, a second series was designed containing the Trp-cage scaffold folded into an N-terminal helix. 

The E6-binding motif was successfully grafted into two parent peptides for the creation of ligands exhibited biological activity while preserving the stable, native fold of their scaffolds. 

These helical peptides inhibit the E6 protein of papillomavirus.

 >pdb|1RIJ|A Chain A,
  E6apn1 peptide

  XLQELLGQWLKDGGPSSGRPPPS

Liu Y, Liu Z, Androphy E, Chen J, Baleja JD. Design and characterization of helical peptides that inhibit the E6 protein of papillomavirus. Biochemistry. 2004 Jun 15;43(23):7421-31. doi: 10.1021/bi049552a. PMID: 15182185.

 

[3] Trp-cage motif

Baru et al. synthesized Trp-cage mutants and studied the influence of folded structures on stability and melting points using NMR. The stabilized construct TC10b was selected for extensive structural and folding thermodynamics evaluation. To study the potential of pH effects, further mutations were performed with Asn, rather than Asp, as the N-cap. 

The studies revealed the the Trp-cage is an 18-residue protein or peptide motif.

The mutational study suggests that indole-backbone interactions rather than specific indole-proline ring interactions appear to be the key component with the high proline content of the Trp-cage improving fold stability.

The Trp-cage motif is an ideal model for exploring the correlation between protein folding simulations and experimental data. The system displays a sharp melting transition, and the Trp-cage is locally structured which makes this a protein of interest to the protein folding research community.  

 

 >pdb|2JOF|A Chain A, TRP-CAGE

  DAYAQWLKDGGPSSGRPPPS

Barua B, Lin JC, Williams VD, Kummler P, Neidigh JW, Andersen NH. The Trp-cage: optimizing the stability of a globular miniprotein. Protein Eng Des Sel. 2008 Mar;21(3):171-85. doi: 10.1093/protein/gzm082. Epub 2008 Jan 18. PMID: 18203802; PMCID: PMC3166533. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166533/

 

[4] Cyclo-TC1 peptide:

The peptide cyclo-TC1 is one of the most stable Trp-cage constructs reported. Scian et al. observed a cyclization-induced ΔΔGU ≥ 8 kJ/mol. Scian et al. reported the X-ray structure for two crystals of a cyclized Trp-cage. The structures observed in the crystal, in the solid state, were quite like the structures observed using NMR, in the solution-state. The research group used automated Fmoc-based solid phase peptide synthesis to produce the peptide. A folding-mediated cyclization of the fully deprotected peptide yielded the cyclic peptide at approximately 10%. The cyclic peptide was obtained with incubating the peptide in 0.5 mM 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide hydrochloride (EDC·HCl), 75 μM N-hydroxy-sulfo-succinimide (Sulfo-NHS) sodium salt at room temperature over a four-day period at a moderately high dilution (50 μM peptide in 25 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 6.5). In the preparative scale reactions, a quench with 2-mercaptoethanol (5 mM) and hydroxylamine·HCl (150 μM) was essential to prevent Trp-cage oligomerization during the reaction work up. 

 >pdb|2LL5|A Chain A, Cyclo-TC1

  GDAYAQWLADGGPSSGRPPPSG

 

Scian M, Lin JC, Le Trong I, Makhatadze GI, Stenkamp RE, Andersen NH. Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12521-5. doi: 10.1073/pnas.1121421109. Epub 2012 Jul 16. PMID: 22802678; PMCID: PMC3411959. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411959/

 

[5] Trp-cage mini protein:

Circular permutation of similar peptide sequences generates small-folded proteins with the same structural motif.

Circular permutation of a protein structure refers to linking the N- and C-terminal ends together by an amide bond or a short peptide linker and cutting out amino acid residues at other places of the peptide sequence. The goal is to alter the sequence of the peptide or mini protein without altering the protein fold.

Byrne et al. studied the Trp-cage fold using circular permutation to observe that a hydrophobic staple near the chain termini is required for enhanced fold stability of the fold topology.

 >pdb|2M7D|A Chain A, Trp-Cage
  mini-protein

  DAYAQWLADXGWASXRPPPS

Byrne A, Kier BL, Williams DV, Scian M, Andersen NH. Circular Permutation of the Trp-cage: Fold Rescue upon Addition of a Hydrophobic Staple. RSC Adv. 2013 Nov 21;2013(43):10.1039/C3RA43674H. doi: 10.1039/C3RA43674H. PMID: 24376912; PMCID: PMC3870897.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870897/

 

[6] Exendrin-4 analogs:

Rovo et al. selected the peptide exendrin-4 (Ex4), a potent glucagon-like peptide-1 receptor agonist involved in regulating the plasma glucose level of patients suffering from type 2 diabetes, as the starting molecule for the rational design of Ex4 analogs with better solubility and a lower tendency for aggregation to minimize side effects in future drugs. The rational design selected started from the parent 20-amino acid, well-folded Trp cage (TC) mini protein. The step-by-step N-terminal elongation of the TC head resulted in the 39-amino acid Ex4 analogue, E19.

 

 >pdb|2MJ9|A Chain A, Exendin-4

  HGEGTFTSDLSKQMEEEXVRLYIQWLKEGGPSSGRPPPS

Rovó P, Farkas V, Stráner P, Szabó M, Jermendy A, Hegyi O, Tóth GK, Perczel A. Rational design of α-helix-stabilized exendin-4 analogues. Biochemistry. 2014 Jun 10;53(22):3540-52. doi: 10.1021/bi500033c. Epub 2014 May 28. PMID: 24828921.  https://pubmed.ncbi.nlm.nih.gov/24828921/

 

[7] A modified Trp-cage:

Graham et al. studied stabilizing interactions in the Trp-cage folded state of the miniprotein. The study observed that a number of stabilizing interactions in the Trp-cage folded state show a strong pH dependence. Carboxylate-protonating conditions in Trp-cage mutants destabilized the fold.

Observed 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.

Graham et al. prepared Trp-cage species that are significantly more stable at pH 2.5 and quantitated the contribution of each interaction listed above. The Trp-cage structure remained constant with the pH change. Additional findings were 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.

 >pdb|6D37|A Chain A,
 
ALA-TYR-ALA-GLN-TRP-LEU-ALA-ASP-DAL-GLY-
         PRO-ALA-SER-DAL-NVA-PRO-PRO-PRO-SER

  XAYAQWLADXGPASXXPPPSX

Graham KA, Byrne A, Son R, Andersen NH. Reversing the typical pH stability profile of the Trp-cage. Biopolymers. 2019 Mar;110(3):e23260. doi: 10.1002/bip.23260. Epub 2019 Feb 19. PMID: 30779444. https://pubmed.ncbi.nlm.nih.gov/30779444/

 The software Chimera from UCSF was used for the illustrations of the structural models.

Alignment of Trp-cage peptide sequences



Additional References


1. Qiu L, Pabit SA, Roitberg AE, Hagen SJ; (2002) Smaller and faster: the 20-residue Trp-cage protein folds in 4 micros. J. Am. Chem. Soc. 124: 12952–12953. [PubMed]

2. Streicher WW, Makhatadze GI (2007) Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy. Biochemistry. 46: 2876–2880. [PubMed]

3. Kannan S, Zacharias M (2014) Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations. PLoS ONE 9(2): e88383. 
https://doi.org/10.1371/journal.pone.0088383https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088383 

4. Jaenicke R. Protein stability and protein folding. Ciba Found Symp. 1991;161:206-16; discussion 217-21. PMID: 1814693. https://pubmed.ncbi.nlm.nih.gov/1814693/


5. Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33-45. doi: 10.1038/355033a0. PMID: 1731198.  https://pubmed.ncbi.nlm.nih.gov/1731198/


6. Lilie H, Lang K, Rudolph R, Buchner J. Prolyl isomerases catalyze antibody folding in vitro. Protein Sci. 1993 Sep;2(9):1490-6. doi: 10.1002/pro.5560020913. PMID: 8104614; PMCID: PMC2142458. https://pubmed.ncbi.nlm.nih.gov/8104614/


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