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

Gabriela Flores-Ramírez,Joel Osuna,Manuel Rivera,Xavier Soberón,Paul Gaytán,Alfredo Morales-Pablos

doi:10.1186/1472-6769-7-1

Gabriela Flores-Ramírez, Joel Osuna + Show 4 more

Open Access

https://doi.org/10.1186/1472-6769-7-1

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Abstract

BackgroundThe effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.ResultsIn 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.ConclusionThe 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.

Highlights

The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties
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 [1,2,3]
We selected the region located between residues 129–142 as target of the mutagenesis for three reasons: 1) It is the longest loop of the protein; 2) two previous attempts of deletions in this area failed to produce fluorescent proteins [10,11]; 3) Published sequence alignments of GFP versus GFP-like proteins of anthozoas suggest that GFP may tolerate deletion of either G138 [16] or H139 [17] (G139 and H140 respectively in superglo GFP (sgGFP))

Summary

Introduction

The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. 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 (page number not for citation purposes). Because of the simplicity of the chromophore formation, modifications on the primary structure of GFP have produced several improved variants, either more fluorescent [4] or blue/red-shifted [5]. These changes have been achieved employing site-directed approaches [6], regional combinatorial approaches [4] and fully random approaches such as DNA shuffling [7]

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