Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein

Abstract

Aequorea victoria green fluorescent protein and its widely used mutants enhanced green fluorescent protein and enhanced cyan fluorescent protein (ECFP) are ideal target proteins to study protein folding. The spectral signals of their chromophores are directly correlated with the folding status of the surrounding protein matrix. Previous studies revealed that tryptophan at position 57 (Trp57) plays a crucial role for the green fluorescent protein's structural and functional integrity. To precisely dissect its role in ECFP folding, we performed its substitution with the isosteric analogs 4-azatryptophan [(4-Aza)Trp] and 7-azatryptophan [(7-Aza)Trp]. Although Trp is moderately hydrophobic, these isosteric analogs are hydrophilic, which makes them an almost ideal tool to study the role of Trp57 in ECFP folding. We achieved high-level expression of both (4-Aza)Trp-ECFP and (7-Aza)Trp-ECFP. However, great portions (70-90%) of protein samples were insoluble and did not contain a maturated chromophore. All attempts to refold the insoluble protein fractions failed. Nevertheless, low amounts of fully labeled, soluble, chromophore containing fractions with altered spectral features were also isolated and identified. The most probable reason for the high yield of misfolding is the introduction of strong hydrophilicity at position 57 which strongly interferes with productive and efficient folding of ECFP. In addition, the results support a strong correlation between translational kinetics of non-canonical amino acids in the ribosome and in vivo folding of the related modified protein sequence.

Probing water micro-solvation in proteins by water catalysed proton-transfer tautomerism

“…In water, however, the N 7 -H tautomer green emission is virtually nonobservable 25 ; this lack has been attributed to the much slower proton-transfer rate constant (B10 9 s À 1 ) together with dominant radiation-less deactivation pathways 22 . The photophysical properties of (7-aza)Trp are very similar to those of 7-azaindole, exhibiting essentially no N 7 -H tautomer form emission 16,[26][27][28] and thus cannot be used for probing any water-associated photophysical phenomena in proteins.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…Similar observations have been made for the increased hydrophilicity after the incorporation of a single azatryptophan into a model protein. 153 In addition, CNBr/formic acid reaction of Tfm containing lysozyme yielded only non-digested protein at Tfm positions. 154 In addition to Tfm, difluoromethionine (Dfm) was incorporated into LAL and studied by 19 F NMR.…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…As an alternative to the directed evolution approach, Budisa et al [42][43][44][45][46] extensively explored a rational approach of addressing the issue of reduced protein folding and stability upon residuespecific incorporation of NCAA (Fig. 3).…”

“…Although X-ray crystal structures of barstar protein with or without substitution of Trp with 4-aminiotryptophan are very similar, the stability of barstar containing 4-aminotryptophan was lower than that of the wild-type barstar due to polar side chain insertion at the hydrophobic protein core [45]. Similarly, replacing Trp at position 57 of enhanced cyan fluorescent protein (ECFP) with either 4-azatryptophan or (3) led to the formation of predominantly insoluble ECFP with immature chromophores; this was attributed to the introduction of strong hydrophilicity at position 57, which interfered with efficient ECFP folding [42]. Methionine oxidation has been implicated in misfolding and aggregation of cellular prion protein (PrP c ) [47].…”