Importance of the carboxyl terminus in the folding and function of alpha-hemolysin of Staphylococcus aureus.
The physical state of two model mutants of alpha-hemolysin (alphaHL), alphaHL(1-289), a carboxyl-terminal deletion mutant (CDM), and alphaHL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native alphaHL. Denatured alphaHL can be refolded efficiently with nearly total recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have revealed the importance of an intact carboxyl terminus in the folding of alphaHL. The CDM exhibits a marked increase in susceptibility to proteases as compared with alphaHL. alphaHL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red-shifted by 9 nm, which indicates a greater solvent exposure of the tryptophan residues of the CDM. In addition, the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike alphaHL and CEM, which show marginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly diminished as compared with alphaHL. These data show that the CDM has several of the features that characterize a molten globule state. Experiments with freshly translated mutants, using coupled in vitro transcription and translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the water-soluble form of alphaHL. The studies demonstrate a critical role of the carboxyl terminus of alphaHL in attaining the native folded state.
Sangha
N
N
National Center for Cell Science, Ganeshkhind, Pune-411007, India.
Kaur
S
S
Sharma
V
V
Krishnasastry
M V
MV
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D009498:Neurotoxins; C021091:staphylococcal alpha-toxin