The effect of amino acid deletions and substitutions in the longest loop of GFP

In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.The green fluorescent protein (GFP) has revolutionized molecular and cell biology, because it can be used as a reporter of gene expression and protein localization due to its inherent capacity to generate an efficiently emitting internal fluorophore [1-3]. GFP is a 28 kDa protein composed of 238 amino acid residues. X-ray crystal studies of GFP uncovered a β-barrel structure resembling a soda can. The wall of the β-can structure is built by 11 antiparallel β-strands. This β-sheet secondary structure surrounds a single central α-helix that contains the fluorophore, spontaneously formed by post-translational modification of residues Ser65, Tyr66 and Gly67. Two protein lids, composed mainly of residues 74–91 and 128–145, cover the β-can structure and isolate the chromophore from the surrounding solvent. Because of the simplicity of the chromophore formation, modi