1. Material and Methods Mutation and transformation: The pMML-LNRA vector contains the wild-type hN1 LNRA gene fused to modified gene that codes for the TrpLE sequence in which the Met and Cys residues have been replaced by Leu and Ala, respectively. The plasmid for hN1LNRA_SD (mutant) was made from the kanamyacin resistant pMML vector for hN1LNRA_WT via QuikChange© Site Directed Mutagenesis (Strategene) and verified via DNA sequencing. Both wildtype and mutant plasmids were transformed into BL21DE3PlysS E.Coli cell line (chloramphenicol resistant) and plated on LB Agar plates containing kanamyacin (50  g/ml) and chloramphenicol (34  g/ml). A single bacterial colony was isolated and grown in LB Broth containing the double antibiotic at 37 °C overnight. This culture was used to prepare 50% glycerol stock solutions and stored at -80 °C. Protein Expression and Purification: hN1LNRA_WT and hN1LNRA_SD were expressed by growing transformed cells to A 600 ~0.6 at 37 °C in a shaking incubator at 220 rpm and inducing protein production with 0.6 mM IPTG. The highly hydrophobic TrpLE sequence in the chimeric protein directs the expressed protein to inclusion bodies. The proteins were purified from inclusion bodies through successive resuspension/ centrifugation steps and and cleaved by cyanogen bromide to obtain hN1LNRA_WT or hN1LNRA_SD. The target protein was purified via reversed-phase HPLC, and lyophilized. The identity of the constructs were confirmed using MALDI-TOF mass spectrometry. Metal Specificity Experiments: Lyophilized protein was resuspended in water containing 20 mM DTT to a concentration of approximately ~110  M. 0.5 mL samples of each construct was dialyzed twice in pre-treated 2000 MWCO micro-dialysis cassettes (Pierce) or dialysis bags (Spectrapor) against 500 mls one of the following nine solutions with a change into a fresh buffer after the first 12 hours. Each sample was dialyzed against a “clean” refolding solution without any cystine/cysteine for an additional 12 hours Protein folding was monitored using a Waters analytical RP-HPLC system running a linear water- acetonitrile (AcN) gradient established by two buffers: Buffer A (10% AcN, 90% H 2 O, 0.1%TFA), Buffer B (90% AcN, 10% H 2 O, 0,1% TFA) The Effect of Various Divalent Cations on the Folding of LNRs Lauren Choi, Wellesley College Advisor: Dr. Didem Vardar-Ulu Introduction Notch receptors are multidomain transmembrane proteins that are important for cell-cell communication and development. Notch receptors have a highly modular architecture (Figure 1). In their extracellular region, they contain a ligand binding domain followed by a Negative Regulatory Region (NRR), where three contiguous Lin-12/Notch Repeats (LNR) wrap around the HD domain containing the regulatory cleavage site (S2) (Figure 2). In the resting conformation of the receptor, these LNRs are critical for masking this regulatory site and prevent activation prior to ligand binding. Correct folding of the LNRs in the receptor is believed to be critical for proper regulation of the Notch signaling pathway. Deregulated notch signaling has been linked to many human diseases such as sclerosis, artereopathy and leukemia. Each LNR is a small 35 residue protein module that contains six cysteines which form three specific disulfide bonds. LNRs also contain a special arrangement of several acidic residues that allow for the coordination of a single Ca 2+ ion, required for folding. (Figure 3). The coordination of the Ca 2+ cation is believed to bring specific cysteine residues into close proximity and facilitate the formation of the correct disulfide bonds. However, it is not known whether other divalent atoms can substitute for Ca 2+ and what minimum concentration of Ca 2+ is sufficient to drive folding. The present study tests the metal specificity of the first wild-type LNR (LNRA_wt) from human Notch1 and a mutated LNRA (LNRA S  D) where serine at position 19 is changed into an aspartic acid, which is the amino acid found at this position in most other LNR sequences. It is hypothesized that this mutation may provide additional coordination sites for the cation during protein folding and hence might impact both metal ion selectivity and binding affinity. To test these hypothesis, unfolded LNRA_wt and LNRA S  D were exposed to identical refolding conditions that contained various cations (Ca 2+, Mg 2+, Zn 2+ and Tb 3+ ) at two different concentrations. The results of this study will help to elucidate the mechanism of metal binding and importance of metal type and concentration during the protein folding process. Results References 1)Gordon, W. R.;* Vardar-Ulu, D.;* Histen, G.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “Structural basis for autoinhibition of Notch” Nat Struct Mol Biol. 2007, 14, 295–300.2. 2) Vardar, D.; North, C. L.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “NMR Structure of a Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067. Future Directions Test the minimum concentration of Ca 2+ necessary for complete correct folding of both LNRA_wt and LNRA S  D constructs. Test additional metals for their effect on the folding of both hN1LNRA_WT and hN1LNRA S  D. Design other mutants that alter binding specificity of hN1LNRA. Isolation and characterization of the mutant species folded in Tb 2+. Folding of LNRA S  D with different cations in the refolding buffer LNRA Folding in Tb 3+ LNRA Folding in Ca 2+ 20mM Tris pH 8.0–8.5 100 mM NaCl 2.5mM cysteine 0.5mM cystine + plus one of the following: Control (no ions) Ca 2+ (1mM or 10mM) Zn 2+ (1mM or 10mM) Mg 2+ (1mM and 10mM) Tb 2+ (1mM and 100uM) 10 20 30 hN1LNRA_WT: EEACELPECQ EDAGNKVCSL QCNNHACGWD GGDCS hN1LNRA_SD: EEACELPECQ EDAGNKVCDL QCNNHACGWD GGDCS Figure 1. Notch Receptor and the Lin12/ Notch Repeats (LNRs). (A) The Domain Organization of the Notch receptor. The negative regulatory region (NRR) is circled. (B) The crystal structure of NRR from Human Notch 2 (1). Note that the LNR modules surround the S2 cleavage site in the resting conformation. (C) The NMR solution structure of LNRA from Human Notch 1 (2). The three disulfide bonds are highlighted in orange and the Ca 2+ coordinating residues are marked in red (aspartate) and green (asparagine). The single serine to aspartate mutation at point 19 is marked in pink. (D) The sequence of LNRA_wt and LNRA S  D from human Notch1. Cysteines are in orange, and the Ca 2+ coordinating residues are highlighted. Ca 2+ LNRA LNRB LNRC BC A Ligand Binding Domain Intracellular Notch N C N15 D33 D30 C4 C9 C22 C27 C18 C34 N-term C-term Ca 2+ S2 A. C. B. D. Conclusions Results of studies using hN1LNRA_WT: Ca 2+ successfully drives proper folding of the wild type LNRA module both at high (10mM) and low (1mM) concentrations indicating that correct folding doesn’t significantly depend on Ca 2+ concentration. Refolding in Mg 2+, Zn 2+ and Tb 3+ do not drive correct folding, but instead result in several misfolded species that resemble the folding in refolding buffer with no multivalent cations. High concentrations (1mM) of Terbium causes complete aggregation of protein while lower concentrations of Terbium have no effect. Results of studies using hN1LNRA_SD: Ca 2+ drives correct folding of the mutant LNRA S  D module both at high (10mM) and low (1mM) concentrations, but folding in Ca 2+ is not as complete as seen for the wild type. Refolding in Mg 2+ and Zn 2+ do not drive correct folding, but instead result in several misfolded species that resemble the folding in refolding buffer with no multivalent cations. Both high (1 mM) and low (100 μM) concentrations of Terbium drive a specific folding event, resulting in an alternatively folded species. Folding of LNRA_wt with different cations in the refolding buffer Figure 2. Chromatograms of hN1LNRA_WT in its reduced form (black) or refolded with no ion (blue), 1 mM Ca 2+ (red), 1 mM Mg 2+ (orange), 1mM Zn2+ (green), 1mM Tb 3 + (purple). Correctly folded LNRA_wt elute earlier in the gradient as seen in the predominant peak (20.5 min.) from 1 mM Ca 2+ folding. The proteins folded with no ion (blue) contain many misfolded species that represent distinct pairs of wrong disulfide bonds and elute as many unresolved peaks. Proteins folded in Mg 2+, Zn 2+ and Tb 3+ result in misfolded species. LNRA_WT fully reduced in TCEP has the highest retention time and shows that all the peaks seen in other chromatograms are different disulfide bonded forms of the same protein. Figure 3. Chromatograms of hN1LNRA_SD in its reduced form (black) or folded with no ions (blue), 1mM Ca 2+ (red), 1mM Mg 2+ (orange), 1mM Zn 2+ (green), 1mM Tb 3+ (purple). 1mM Ca 2+ folding shows incomplete correct folding of the mutant protein. Mg 2+ and Zn 2+ result in misfolded species identical to the protein folded with no ions. Tb 3+ causes in the formation of an alternatively folded species. Figure 4. Chromatograms of hN1LNRA_WT and hN1LNRA_SD folded in 1 mM or 10 mM Ca 2+. The folding of LNRA_WT (10 mM red, 1 mM orange) and LNRA S  D (10 mM black, 1 mM blue) exhibit no significant dependency on Ca 2+ concentration. The peak at 20 minutes represent the properly folded species. Note: The shift in the elution time for LNRA S  D in 10 mM Ca 2+ with respect to the others is due to a slight change in the buffer conditions used for that run. Figure 5. Chromatograms of hN1LNRA_WT and hN1LNRA_SD folded in 1 mM or 10 mM Tb 3+. The folding of the alternatively folded species of the LNRA S  D exhibits no significant dependence on Tb 3+ concentration (10mM black, 1mM blue). LNRA_wt folded at 10mM Tb 3+ (red) elutes only after 100% Buffer B wash indicating aggregated species whereas LNRA_wt folded at 1mM Tb 3+ (orange) give similar results to folding with no ion (Figure 2).