Protein design four-helix bundles

Other approaches to de novo four-helix bundle proteins have emphasized nonrepetitive designs. One such example is the four-helix bundle protein Felix (53), a 79-residue protein which uses 19 of the 20 naturally occurring amino acids ... 

DeGrado, W.F., Regan, L., Ho, S.P. The design of a four-helix bundle protein. Cold Spring Harbor Symp. Quant. Biol. 52 521-526, 1987. 

Designed histidine-based four-helix bundle proteins have been shown to catalyze the reactions ofp-nitrophenyl esters [13]. The reactivity of histidine is due to its imidazoyl side chain that reacts with active esters in a two-step reaction. In the first and rate-limiting step the imidazoyl residue reacts with the ester to form an acyl intermediate under the release of p-nitrophenol and in the second step the acyl intermediate reacts with the most potent nucleophile to form the reaction products. 

Fig. 17. An incremental approach to the design of a four-helix bundle protein (Ho and DeGrado, 1987). (a) The sequence of a peptide is first optimized for forming a very stable tetramer of a helices. The stability of the tetramer can be assessed from the dissociation constant for the cooperative monomer-to-tetramer equilibrium, (b) Two optimized helical sequences are then connected in a head-to-tail manner by a single loop. The loop sequence can be optimized by evaluating a series of alternate designs, (c) Finally, the entire four-helix bundle structure can be constructed from four optimized helices and three optimized loops.

Figure 5.1. Modelled structure of a 42-residue peptide folded into a helix-loop-helix motif and dimerized to form a four-helix bundle protein. Helices are amphiphilic with a hydrophobic and a polar face. Due to the robustness and ease of synthesis this has become a popular motif in de novo protein design.

Hecht M.H. Richardson. J.S. Richardson. D.C. Ogden, R.C. De novo design, expression, and characterization of Felix A four-helix bundle protein of native-like sequence. Science 1990. 249. 884-891. 

They started from the sequence of a domain, Bl, from an IgG-binding protein called Protein G. This domain of 56 amino acid residues folds into a four-stranded p sheet and one a helix (Figure 17.16). Their aim was to convert this structure into an all a-helical structure similar to that of Rop (see Chapter 3). Each subunit of Rop is 63 amino acids long and folds into two a helices connected by a short loop. The last seven residues are unstructured and were not considered in the design procedure. Two subunits of Rop form a four-helix bundle (Figure 17.16). 

Helical heptad repeat sequences have been reported to be well behaved although they are difficult to characterize by NMR spectroscopy due to spectral overlap. The motifs that have been shown to have native-like properties, and are not highly repetitive, have cores composed of aromatic amino acid side chains of, for example, phenylalanine and tryptophan. In four-helix bundle motifs [1, 2], the /1/la-motif BBAl [5] and the /1-sheet protein Betanova [9], the formation of the folded structure appears to be strongly dependent on such residues although the energetics have not been calculated by substitution studies. As a tentative rule, therefore, the probability of success in the design of a new protein is probably much higher if residues are included that can form aromatic clusters in the core (Fig. 5). 

In an alternative approach the site-selective functionalization reaction has been used to incorporate a galactose derivative into a folded foux-hehx bundle protein and the effect of glycosylation on the structure of the folded protein has been identified [65]. The unfunctionalized designed four-helix bundle did not have a well-defined tertiary structure but the introduction of the sugar improved the helical content and reduced the rate of conformational exchange. Glycosylation may therefore play a role in the maturation of poorly folded proteins. 

Since the primary structure of a peptide determines the global fold of any protein, the amino acid sequence of a heme protein not only provides the ligands, but also establishes the heme environmental factors such as solvent and ion accessibility and local dielectric. The prevalent secondary structure element found in heme protein architectures is the a-helix however, it should be noted that p-sheet heme proteins are also known, such as the nitrophorin from Rhodnius prolixus (71) and flavocytochrome cellobiose dehydrogenase from Phanerochaete chrys-osporium (72). However, for the purpose of this review, we focus on the structures of cytochromes 6562 (73) and c (74) shown in Fig. 2, which are four-a-helix bundle protein architectures and lend themselves as resource structures for the development of de novo designs. 

Fig. 16. Schematic representation of the assembly of designed heme protein SAMs on silanized quartz substrates. Designed peptides are synthesized, homodimerized, and selfassociate to form four-helix bundles prior to heme incorporation, followed by chemisorption on prepared quartz surfaces. Reprinted with permission from Ref. (185) copyright 1998 American Chemical Society.

K.H. Lee, H.Y. Lee, M.M. Slutsky, J.T. Anderson, E.N.G. Marsh, Fluorous effect in proteins De novo design and characterization of a four-ot-helix bundle protein containing hexafluoroleucine. Biochemistry 43(51) (2004) 16277-16248. 

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