title: Alkaline transition of pseudoazurin Met16X mutant proteins: protein stability influenced by the substitution of Met16 in the second sphere coordination.
abstract: Several blue copper proteins are known to change the active site structure at alkaline pH (alkaline transition). Spectroscopic studies of Met16Phe, Met16Tyr, Met16Trp, and Met16Val pseudoazurin variants were performed to investigate the second sphere role through alkaline transition. The visible electronic absorption and resonance Raman spectra of Met16Phe, Met16Tyr, and Met16Trp variants showed the increasing of axial component at pH approximately 11 like wild-type PAz. The visible electronic absorption and far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at pH>11. Resonance Raman (RR) spectra of PAz showed that the intensity-weighted averaged Cu-S(Cys) stretching frequency was shifted to higher frequency region at pH approximately 11. The higher frequency shift of Cu-S(Cys) bond is implied the stronger Cu-S(Cys) bond at alkaline transition pH approximately 11. The visible electronic absorption and far-UV CD spectra of Met16X PAz revealed that the Met16Val variant is denatured at pH>11, but Met16Phe, Met16Tyr, and Met16Trp mutant proteins are not denatured even at pH>11. These observations suggest that Met16 is important to maintain the protein structure through the possible weak interaction between methionine -SCH3 part and coordinated histidine imidazole moiety. The introduction of pi-pi interaction in the second coordination sphere may be contributed to the enhancement of protein structure stability.
authors.affiliation: Institute of Applied Beam Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Rehab F
authors.initials: RF
authors.lastname: Abdelhamid
authors.forename: Yuji
authors.initials: Y
authors.lastname: Obara
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Kohzuma
mesh_terms: D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites / Q000187:drug effects*; D002942:Circular Dichroism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: C047163:pseudoazurin; D001400:Azurin; D008715:Methionine