Saint Mary’s College of California Summer Research Program 2010 1 Thimet Oligopeptidase: Fluorescent Labeling to Investigate Structural Changes Gabrielle B. Diaz, and Jeffrey A. Sigman. Chemistry Department, Saint Mary’s College of California, 1928 St. Mary’s Road, Moraga, CA 94556, U.S.A. The intent of the present study was to optimize and fractions were collected; currently they are awaiting procedures to monitor structural changes in the enzyme mass spectrometry analysis at a facility in UCSF. Once the thimet oligopeptidase (TOP). Wild-type rat TOP was cova- location of attachment is identified by mass spectrometry, lently labeled with the cysteine-specific, fluorescent probe further assays will be done to analyze the sensitivity of the N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-1,3-di- fluorophore on site. Depending on the results, the enzyme azol-4-yl)ethylenediamine (IANBD). Adding this fluorescent will be mutated to rearrange the site of IANBD attachment tag is analogous to adding an antennae for detection. This to more plausible sensitive sites. will allow structural changes to be monitored and analyzed Activity assays were done and confirmed that the under fluorescence spectroscopy due to the tag’s sensitiv- modification did not affect enzymatic performance. Ligand ity to the polarity of its environment. binding assays were also done with the inhibitor angio- The procedure done to modify the enzyme with tensin (1-7) to detect initial changes in fluorescence when IANBD involved covering surface cysteines with iodo- the enzyme is open or closed. acetate, leaving cysteines within the substrate binding Conducting this research will provide more knowl- cleft available for IANBD attachment. A tryptic digest and edge on the structure and function of TOP in an outcome HPLC was done to isolate the peptide segment contain- to provide drugs and inhibitors that will bind strongly to ing the fluorophore. The peptide fragments were eluted the enzyme. 256 J. A. Sigman and others INTRODUCT I ON Recombinant TOP preparation Thimet oligopeptidase (TOP) (EC 3.4.24.15) is a TOP (accession number P24155) was expressed and purified as metallo endopeptidase that is ubiquitous throughout described previously [15]. The enzyme concentration was deter- 1 the body with concentrated presence in the brain and mined using the molar absorption coefficient ε280 73.11 mM− 1 = · cm− , calculated based on the amino acid content of the protein gonads. It is homologous to several metallopeptidases using the automated ProtParam Tool on the SWISS ExPASyserver across species of which all share a common HEXXH [5,16]. metal-binding motif1-2 i.e., a recurring structure. TOP functions to cleave an array of peptides which in Kinetic assays effect involve immune responses and regulation Kinetic assays were performed using a Cary Eclipse spectro- of physiological activities such as lowering blood fluorimeter or PerkinElmer luminescence spectrometer LS 50 B. 1-2 The cleavage of the fluorogenic MCA [17] or mca-Bk substrate pressure and reproduction . Another function that was monitored by the increase in emission at 400 nm over time has intrigued further research is the enzyme’s ability using an λexcitation of 325 nm. Substrate concentration was calcu- 1 lated based on the molar absorption coefficient ε365 (17.3 mM− to degrade Aβ amyloid plaques present in Alzheimer’s 1 · cm− ) of the DNP. Product formation was determined to be linear 3 disease . with time under all conditions monitored, and less than 10% of the substrate was consumed during the assay. Assays were X-ray diffraction studies have provided performed in duplicate at 23 ◦C in 25 mM Tris/HCl at pH 7.8, invaluable information with regards to its structure adjusted to a conductivity of 12 mS/cm2 with KCl and containing and confirmed structural homology to its closest 10% glycerol. The temperature was chosen so as to minimize relative, neurolysin1-2. Figure 1 shows the overall breakdown of both enzyme and substrate and to allow for sensi- tivity at low substrate concentrations; the pH represents the opti- structure; TOP is primarily α-helical with two domains mal value for kcat/Km for MCA [5]. TCEP (1 mM) was also added separated by a deep cleft where the substrate binds. to the buffer to prevent protein dimerization [29]. For assays in urea, the above buffer and an identical buffer containing approx. Domain II contains the Zn(II) active-site and residues 1 FigureFigure 1 1: Overall Tertiary tertiary structure structure of TOP showing of TOP, the indicating location ofthe the domains. active site 10 M urea were mixed in the appropriate ratio. The final urea zinc (white sphere) and the tryptophan residues (space filled) present in concentration was determined based on the refractive index of domains I and II the solution [18]. The change in fluorescence intensity over time was converted into rate of product formation using a standard curve calculated for the peptide products. Individual stan- TOP contains seven tryptophan residues distributed unevenly be- dard curves were prepared at each urea concentration. Although tween the two domains. Trp335 and Trp614 reside in domain I, and the intercept of the standard curve changed, the slope was found to tryptophan at positions 26, 124, 389, 511 and 513 are found in be independent of urea concentration. The kinetic parameters V max domain II. We here report on use of the denaturing agent urea and Km were determined using a hyperbolic fit rate V max[S]/ { = as a tool to study partial unfolding of TOP, as monitored by fluor- (Km [S]) to the plot of substrate concentration (µM) versus escence emission, collisional quenching and anisotropy. Unfold- rate+ of product} formation (µmol/s per µmol of enzyme) under ing occurs as a two-step process, with loss of the catalytic zinc conditions in which [S] is above and below Km. occurring only with the second unfolding event. Complete loss of activity towards a 5-residue quenched fluorescent substrate MCA (7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-Lys-di- HPLC analysis nitrophenol) is apparent at even low urea concentrations, but activ- Products of the enzymatic reactions with mca-Bk were analysed ity towards a 9-residue Bk-derived substrate is enhanced at low by HPLC on a Hewlett Packard 1090 apparatus. The reaction urea levels. These consistent results show that TOP undergoes mixture, 200 µl total volume in Tris buffer or Tris buffer with conformational changes that differentially affect substrate recog- 2 M urea, contained mca-Bk (0.4 mg/ml) and 0.05 µM enzyme. nition, and suggest a mechanism by which TOP can accommodate A sample was taken at 0 min (before initiation of the reaction) and substrate variation. after reacting for 30 min at 23 ◦C. The reaction was terminated with an equal volume of 1% trifluoroacetic acid in methanol. A 20-µl aliquot was subjected to reverse-phase HPLC using EXPERIMENTAL aC183µ column (150 mm 4.6 mm; Alltech). Solvent A was acetonitrile and solvent B 0.1×% trifluoroacetic acid in water. A Materials linear gradient of 10–82% solvent A was applied, and the pro- Glutathione–Sepharose, Sephacryl S-200 and PD-10 columns ducts were detected by absorbance monitored at 330 nm. were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, U.S.A.). Tris/glycine polyacrylamide gels (12%) were from Invitrogen (Carlsbad, CA, U.S.A.), and stained with Gelcode DSC (differential scanning calorimetry) Blue from Pierce (Rockford, IL, U.S.A.). TCEP [tris-(2-carboxy- All experiments were performed using a VP-DSC microcalori- ethyl)phosphine] was also purchased from Pierce. The quenched meter (MicroCal Inc., Northampton, MA, U.S.A.) at an upscan fluorescent substrates MCA and modified bradykinin, mca- rate of 60 ◦C per h over the range 20◦ to 110 ◦C. Degassed buffer, Bk 7-methoxycoumarin-4-acetyl-[Ala7, Lys(DNP)9]-bradykinin, identical with that diluting the sample, was used as the reference. where{ DNP is 2,4-dinitrophenyl , were obtained from Bachem The injection volume was 0.51 ml. Raw heat data were trans- (King of Prussia, PA, U.S.A.). All} other reagents were purchased formed and plotted as heat capacity as a function of temperature from Sigma Chemical Co (St. Louis, MO, U.S.A.). using the Origin for DSC software supplied by the manufacturer. c 2005 Biochemical Society � Saint Mary’s College of California Summer Research Program 2010 G. B. Diaz 2 necessary for peptide cleavage. Domain I has a are located on the surface5. However, two residues, putative role for limiting substrate accessibility to Cys175 and Cys425, are located within the substrate- the active site1. Between the two domains, a hinge binding cleft and are the target of attachment5. movement has been speculated due to the flexible Tagging the enzyme will demonstrate if the sites are loop regions connecting the domains. This movement sensitive to structural changes when monitored by poses curiosity with regards to substrate recognition, ligand binding assays. If the expected data and results specificity and selectivity which can be connected to are contrary, these two cysteines or other amino acids the function and properties of homologous enzymes.1 can be mutated into other residues to change the site In the present research, a method has been of attachment to a more sensitive location. modified and produced to attach a fluorescent tag to the enzyme and to identify the location of EXP E R I M E NTAL PROC E DUR E S attachment. The purpose of tagging the enzyme is to investigate and monitor structural changes during Materials activity, particularly the hinge movement collaborated Wild-type rat thimet oligopeptidase was by the two domains. provided by M.J. Glucksman (FUHS/Chicago Medical This investigation will lead to better drug School, Midwest Proteome Center and Department design and understanding of enzymatic activity.