Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes that catalyze synthesis of various medically important peptide compounds. The biosynthetic process starts with the adenylation (A) domain, which catalyzes in the first half reaction the synthesis of the aminoacil-adenylate, while in the second half reaction, the activated amino acid is transferred to the adjacent thiolation (T) domain. In this study, the structural dynamics of the A-domain from tyrocidine synthetase 1 were investigated by the fluorescence spectroscopy method. The amino acid sequence of TycA-A has five tryptophan residues: W227, W301, W323, W376 and W406. Quenching of fluorescence of the wild type and mutant TycA-A proteins with acrylamide, revealed two populations of tryptophan residues with a different degree of their side chain accessibility to the surrounding solvent. The presence of substrates (L-Phe and ATP) induced a conformational change in the wild type and all mutant TycA-A proteins, except W227F. The conformational change of the protein, as a consequence of substrate binding, does not occur when tryptophan 227 is substituted with phenylalanine. This leads to the conclusion that the conformational change of the protein is localized in the vicinity of this specific tryptophan residue. Quenching of fluorescence of the TycA-A proteins with potassium iodide, along with an analysis of the quencher efficiency, shows that acrylamide, in comparison with potassium iodide, is a more efficient quencher.