Mini-proteins

Cyclotides, circular mini-proteins bearing a cystine knot motif, are produced by plants as part of their defense mechanism. These cyclic proteins are thermostable and were recently shown to disrupt the membrane of human lymphoma cell line HeLa, causing cellular leakage. The combined approach growth- and non-growth-associated pattern provided the accumulation of 370-mg cyclotide kalata Bl/gdw within Oldenlandia affinis cells in culture. " ... 

Although small in size, the conotoxins contain many of the structural elements present in larger proteins, including a-helices, -sheets and fl-turns, hence, they are often referred to as mini-proteins. Their relative ease of synthesis allows accurate three-dimensional structures to be obtained using techniques such as X-ray crystallography and NMR spectroscopy. 

In addition to incorporating the 4-(2-aminoethyl)dibenzofuran-6-propanoic acid template into small peptides where a reverse turn is desired, we have also recently incorporated this template into a mini-protein called the PIN WW domain. WW domains have a three-stranded antiparallel p-sheet structure that mediates intracellular protein-protein interactions. 31 Substitution of this 3-turn mimetic into loop 1 of the PIN WW domain leads to a folded, three-stranded, antiparallel p-sheet structure with a stability indistinguishable from that of the all a-amino acid sequence. The template-incorporated PIN WW domain (11) was synthesized by an Fmoc-based solid-phase peptide synthesis strategy (Scheme 8), utilizing N-Fmoc-protected 4-(2-aminoethyl)dibenzofuran-6-propanoic acid 10. 11 The synthesis of 10, similar to that of 8, has been published.1 1 ... 

Bottger, A., Bottger, V., Sparks, A., Liu, W.L., Howard, S.F. and Lane, D.P. (1997) Design of a synthetic MDM2-binding mini protein that activates the p53 response in vivo. Curr. Biol., 7, 860-869. 

The seeds of squash plants are rich in a family of trypsin and chymotrypsin inhibitors that are approximately 35 amino acids in size and have been extensively investigated not only for their enzyme inhibitory activity, but also because they are very stable mini-protein scaffolds with applications in protein engineering. The best studied examples are Ecballium elaterium trypsin inhibitor (EETI-II) and Cucurbita maxima trypsin inhibitor (CMTI). Both X-ray and NMR have been used to characterise their structures, which incorporate a cystine-knot motif formed by three conserved disulphide bonds.93 We will describe this motif in more detail in a later section describing the plant cyclotides. 

When bound to the receptor/acceptor, the peptide will be an integral part of the receptor conformation (i.e., behave as a "mini" protein). 

Fig. 13 Stabilized helices and nonnatural helix mimetics several strategies that stabilize the a-helical conformation in peptides or mimic this domain with nonnatural scaffolds have been described. Recent advances include [1-peptide helices, terphenyl helix-mimetics, mini-proteins, peptoid helices, side-chain crosslinked a-helices, and the hydrogen bond surrogate (HBS) derived a-helices. Circles represent amino acid side-chain functionality (Reprinted from Henchey et al. [52], Copyright (2008) with permission from Elsevier)...

Feng, J. W. A., J. Kao, and G. R. Marshall. 2009. A second look at mini-protein stability Analysis of FSD-1 using circular dichroism, differential scanning calorimetry, and simulations. Biophysical Journal. 97, 2803. 

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) use a Mini-Protein II slab-gel system (Bio-Rad) or equivalent. 

Satoh D et al (2006) Folding liee-energy landscape of a 10-residue mini-protein, chignolin. FEES Lett 580 3422-3426... 

T. Yamazaki and A. Kovalenko. Spatial decomposition of solvation free energy based on the 3D integral equation theory of molecular liquid Application to mini proteins. 

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