title: The effect of amino acid deletions and substitutions in the longest loop of GFP.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  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-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Flores-Ramírez
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rivera
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Morales-Pablos
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Joel
authors.initials: J
authors.lastname: Osuna
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Xavier
authors.initials: X
authors.lastname: Soberón
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
authors.forename: Paul
authors.initials: P
authors.lastname: Gaytán