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)

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

“…The staphylococcal nuclease is a relatively small (149 amino acids) nonglycosylated protein devoid of cysteine residues. One of these residues was introduced in position 70 by replacing a lysine (K70C) (38). A high mannose type glycopeptide was then linked to that position using Nsuccinimidyl 3-(2-pyridyldithio) propionate, a bifunctional reagent that reacts with amino and sulfhydryl groups.…”

“…A high mannose type glycopeptide was then linked to that position using Nsuccinimidyl 3-(2-pyridyldithio) propionate, a bifunctional reagent that reacts with amino and sulfhydryl groups. The neoglycoprotein thus formed (K70C-Glyc) had the same specific nuclease activity as the unmodified protein, thus suggesting that both species had the same tertiary structure (38,39). The neoglycoprotein had a very low glucose acceptor capacity but addition of the nuclease inhibitor pdTp [3,5 diphosphothymidine], a stabilizer of the native nuclease conformation and an inducer of proper folding in truncated species (see below), further diminished the glucose acceptor capacity.…”

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

“…Therefore, the a N-terminal domain must contain determinants for dimeriza- [24][25][26][27]; and (iii) we analyze the cleavage products by denaturing PAGE followed by PhosphorImager analysis. Binding of the ligand decreases polypeptidebackbone solvent accessibility at residues it contacts, protecting against hydroxyl-radical-mediated cleavage at residues it contacts and, therefore, resulting in a gap in the "ladder" of cleavage products.…”

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

“…Prominent among these tools is the use of hydroxy radical (•OH) reactions to explore the structure and interactions involving nucleic acids [18][19][20] and proteins [21][22][23][24][25][26][27][28]. However, the •OH species shows some chemical selectivity in its reaction with peptide targets, thus potentially biasing the analysis to particular spots.…”