The effect of amino acid deletions and substitutions in the longest loop of GFPReport as inadecuate




The effect of amino acid deletions and substitutions in the longest loop of GFP - Download this document for free, or read online. Document in PDF available to download.

BMC Chemical Biology

, 7:1

First Online: 26 June 2007Received: 15 February 2007Accepted: 26 June 2007

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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6769-7-1 contains supplementary material, which is available to authorized users.

Gabriela Flores-Ramírez, Manuel Rivera contributed equally to this work.

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

Source: https://link.springer.com/



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