Incorporation of Tryptophan Analogues into Staphylococcal Nuclease:  Stability toward Thermal and Guanidine-HCl Induced Unfolding

• Published in: Biochemistry (Easton) 1998-06, Vol.37 (25), p.8947-8953
• Main Authors: Wong, Cing-Yuen, Eftink, Maurice R
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Circular Dichroism; Enzyme Stability - genetics; Guanidine - chemistry; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - genetics; Micrococcal Nuclease - metabolism; Octanols; Protein Folding; Staphylococcus; Thermodynamics; Tryptophan - analogs & derivatives; Tryptophan - chemistry; Tryptophan - genetics; Tryptophan - metabolism; Valine - genetics; Water
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971863g

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Δ137−149 fragment of the latter mutant. The guanidine−HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Δ137−149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.