title: Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.
abstract: Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387] and used to assess the ability of this cleavage reagent to faithfully report on the structure of nonnative protein states. The act of mutation and modification did not significantly alter the protein's global structure, as measured by CD and enzymatic activity, and only modestly affected its stability. The reaction was conformation dependent and generated specific cleavage products that mapped tertiary interactions present in the folded state. Several parameters relevant to the cleavage reaction and its use as a conformational probe were analyzed. Proximity and solvent accessibility are the most important parameters in determining the cleavage pattern and can be used to predict cleavage sites in the native protein. The cleavage reaction can be performed in the presence of high denaturant concentration, in the presence of SDS, and under a wide range of pH values; thus it can readily be applied to the study of equilibrium folding intermediates. Mass spectrometric analysis combined with N-terminal sequencing identified cleavage products consistent with a single cleavage event per protein molecule and revealed one cleavage mechanism which was not previously considered for protein oxidative degradation, although it was reported for hydroxyl radical induced cleavage of small peptides. Identification of the cleavage sites obtained from each variant allowed a nearest-neighbor mapping of the secondary structural elements of nuclease. Quantitation of specific cleavage products was used to monitor the disruption of the interaction between helices H2 and H3 in equilibrium unfolding experiments. The resulting unfolding curve revealed a local conformational heterogeneity at low denaturant concentration which was not observed when the same transition was monitored by the change in fluorescence of a single nuclease tryptophan. Interestingly, the midpoint of the transition and the second half of the unfolding curve were the same, as monitored by the two probes. This indicates that the lifetime of the reactive oxygen species generated by the cleavage reagent is short compared to the unfolding equilibrium rate constants and that the cleavage technique identifies a native-like folding intermediate not detected by fluorescence. The experiments presented herein demonstrate that EPD-Fe-mediated protein cleavage is an appropriate technique for the study of nonnative protein structure.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520.
authors.forename: M R
authors.initials: MR
authors.lastname: Ermácora
authors.forename: D W
authors.initials: DW
authors.lastname: Ledman
authors.forename: H W
authors.initials: HW
authors.lastname: Hellinga
authors.forename: G W
authors.initials: GW
authors.lastname: Hsu
authors.forename: R O
authors.initials: RO
authors.lastname: Fox
mesh_terms: D003196:Computer Graphics; D003545:Cysteine / Q000737:chemistry*; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D004795:Enzyme Stability; D005069:Evaluation Studies as Topic; D007501:Iron / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: C084719:EDTA-(2-aminoethyl 2-pyridyl disulfide)-Fe; D015335:Molecular Probes; D004492:Edetic Acid; D007501:Iron; D008836:Micrococcal Nuclease; D003545:Cysteine