A thorough conformational search of the chromophore-forming region of immature green fluorescent protein (GFP) revealed that it is preorganized in a unique conformation required for chromophore formation. This "tight turn" conformation has an i carbonyl carbon to i + 2 amide nitrogen distance of less than 2.90 Angstrom with phi = 60 +/- 30 degrees and psi = 30 +/- 15 degrees. Less than 1.00% of the residues of the 50 representative proteins examined adopt this conformation. The tight turn conformation is predominately located on the periphery of the proteins or in flexible areas, except in GFP. Molecular dynamics simulations and Ramachandran plots show the chromophore-forming region in immature GFP can only adopt the tight turn conformational family. Moreover, this conformation is ideally suited for the cyclization necessary for chromophore formation, i.e., for nucleophilic attack of the amino group of Gly67 on the carbonyl group of Ser67. The 11 beta sheets of GFP force the chromophore-forming peptide fragment to adopt a conformation that has an exceptionally short interatomic distance between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 and lock it into this conformation. Several mutant GFPs have been expressed that exhibit greater solubility and thermostability than wild-type GFP. These properties are linked to protein folding and chromophore formation. Our calculations show that the mutations cause a shortening of the distance between the carbonyl carbon of Ser65 and the amide nitrogen df Gly67, and therefore enhance chromophore formation.