1. Material and Methods Cell Growth and Protein Expression: The protein constructs of interest were expressed by transforming BL21(DE3)-pLysS E. coli cells with the kanamycin-resistant pMML-LNRB vector that contained the DNA sequences for the constructs of interest with an N-terminal hydrophobic leader sequence. These mutations were made via QuikChange© Site Directed Mutagenesis and confirmed with DNA sequencing. Inclusion Body Purification and Identification: To dispose of undesired cellular parts, the samples were suspended in different buffers via sonication. After suspension in each buffer, the samples were centrifuged in SS34-rotor tubes at 19K for 30 minutes to separate the proteins from the undesired cellular parts. Buffers Used: Buffer A: 50 mM Tris pH 8.0, 25% Sucrose, 200 mM of NaCl Buffer B: 20 mM Tris pH 8.0, 1% Triton X-100 Buffer C: 20 mM Tris pH 8.0, 200 mM of NaCl Buffer D: 6 M Guanidine HCl, 50 mM Tris pH 8.0, 10 mM DTT The protein modules with the leader sequence still attached were dissolved in  70% formic acid then cleaved with  200 mg cyanogen bromide for 4 hours. The samples were dialyzed overnight in acidic water  pH 3. After the overnight dialysis, the leader sequence was crashed out by raising the pH of the samples to ~8.5. The protein modules were further purified using reversed-phase (RP) prep HPLC. After purification, the identities of the protein modules were confirmed using MALDI-TOF mass spectrometry. HPLC Buffers Used: Buffer A: 10% Acetonitrile, 90% H 2 O, 0.1% TFA Buffer B: 90% Acetonitrile, 10% H 2 O, 0.1% TFA Refolding Tests: Refolding Buffer: 20 mM Tris pH 8.0-8.5,10mM CaCl 2, 100 mM NaCl, 2.5 mM cysteine, 0.5 mM cystine 15  l of  200  M protein was immersed in 285  l of refolding buffer for different lengths of time. Time points used: 0 hr, 1 hr, 1.5 hrs, 3 hrs, 7 hrs, overnight (  16 hours) Folding was quenched using 50  l of 4% TFA. The “folded” samples were run on the RP - analytical HPLC. LIN-12/Notch Repeat B: The effects of disulfide bonding and hydrophobic residues on its autonomous folding The effects of disulfide bonding and hydrophobic residues on its autonomous folding Jessica Lin ‘10, Wellesley College Advisor: Dr. Didem Vardar-Ulu Abstract Results Reference Gordon, W.R., Vardar-Ulu, D., Histen, G., Sanchez-Irizarry, C., Aster, J.C., Blacklow, S.C. (2007) Structural basis for autoinhibition of notch. Nature Structural &Molecular Biology. Discussion and Future Direction: eliminating two cysteines (resulting in four cysteines) in LNRB_original leads to three thermodynamically preferred folded conformations instead of just one preferred conformation, which is seen in the wild type LNRB_original with six cysteines in LNRB_original, changing W52 to a less hydrophobic residue, alanine, does not increase the stability of the autonomous folding of LNRB as was initially hypothesized; after analyzing the results, it is now hypothesized that eliminating W52 actually destabilizes LNRB_original because the stabilizing hydrophobic interactions between W52 and W42 are eliminated as well in LNRB_short, no conclusive effects of eliminating W52 on folding were observed; at the 3-hour refolding time point for mutW52  A52_short, a more defined peak was observed later in the gradient (a more hydrophobic conformation), but the peak was no longer visible in the sample that refolded overnight Future Direction testing the four LNRB_intermediate constructs to see whether the same effects of eliminating two cysteines and mutating W52 to an alanine on folding will be observed optimizing the conditions in which to refold and analyze the LNRB_short constructs in order to obtain more conclusive results Notch proteins are transmembrane proteins involved in controlling cell differentiation, cell growth, and cell death during embryonic and adult life. Upon ligand binding, Notch proteins are activated and a proteolytic cleavage is induced within an extracellular negative regulatory region (NRR) (Gordon et al. 2007). The NRR contains three contiguous LIN-12/Notch Repeats (LNRs) – LNRA, LNRB, and LNRC – the first two of which are currently being studied in this lab to understand more thoroughly the important determinants in protein folding. Previous research has shown that imperative to LNR folding are its calcium ion coordination and correct disulfide bond formation. Disulfide bonds in proteins are covalent bonds formed by two cysteine residues that contribute to the tertiary structure of proteins alongside with other global, non-covalent interactions. To study the effect of each disulfide bond on the independent folding of LNRB, one of its three pairs of disulfide bonds was eliminated by mutating C45 and C69 to two alanine residues. In this study, the mutant form was folded in vitro under the same conditions as the wild type, and the folding patterns of the two protein modules were compared. One other characteristic of much interest in LNRB is its hydrophobic residues and their impact on the autonomous folding of LNRB. Because of its more central location in relation to the rest of the protein, LNRB participates in more extensive interactions via its exposed hydrophobic residues such as W29, W42, and W52. With an indole functional group, tryptophan is one of the most hydrophobic amino acids. Hence, to study the possible destabilizing effect of hydrophobicity in LNRB, W52 was mutated to an alanine. W52 is especially of interest because of its location on the outer surface of LNRB right next to C51 and its proximity to W42, which is located in the LNR-AB linker. It is hypothesized that the hydrophobic interaction between W52 and W42 counteracts the stability imparted by the disulfide bond residing in the core of the LNRB module, formed by C51 and C64. Different lengths of LNRB were tested in conjunction with the above-mentioned mutations in the hopes of defining the minimal requirements for the autonomous folding of LNRB. Protein construct Calculated MW (Da) Observed MW (Da) LNRB_Original L N F N D P W K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q R A E G Q 5368.85357.72 Mut1,5 C  A _LNRB_Original L N F N D P W K N A T Q S L Q C W K Y F S D G H C D S Q C N S A G A L F D G F D C Q R A E G Q 5304.75291.05 Mut W52  A52_LNRB_Original L N F N D P W K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q R A E G Q 5253.7N/A Mut 1,5 C  A_Mut W52  A52 LNRB_Original L N F N D P W K N A T Q S L Q C A K Y F S D G H C D S Q C N S A G A L F D G F D C Q R A E G Q 5189.5N/A LRNB_Intermediate D P W K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q 4338.7N/A Mut 1,5 C  A LNRB_Intermediate D P W K N A T Q S L Q C W K Y F S D G H C D S Q C N S A G A L F D G F D C Q 4274.6N/A Mut W52  A52 LNRB_Intermediate D P W K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q 4223.6N/A Mut 1,5 C  A_Mut W52  A52 LNRB_Intermediate D P W K N A T Q S L Q C A K Y F S D G H C D S Q C N S A G A L F D G F D C Q 4159.4N/A LNRB_Short K N C T Q S L Q C W K Y F S D G H C D S Q C N S A G C L F D G F D C Q 3940.3N/A Mut W52  A52 LNRB_Short K N C T Q S L Q C A K Y F S D G H C D S Q C N S A G C L F D G F D C Q 3825.1N/A Acknowledgments Dr. Didem Vardar-Ulu Other members of the lab: Sharline Madera ’08, Christina Hao ’09, Lauren Choi ’10 Roberta Day Staley and Karl A. Staley Fund for Cancer-Related Research Table 1: LNRB constructs of interests with their molecular weights. The LNR-AB and BC linkers are highlighted in red. The LNRB_intermediate constructs have part of the AB linker and the LNRB_short constructs do not have any part of either linker. The cysteines forming disulfide bonds are highlighted in blue, the C  A mutations are highlighted in orange, and the W  A mutations are highlighted in purple. The MW’s were calculated using ExPASy ProtParam and the observed MW’s were found using a MALDI-TOF mass spectrometer. Several observed MW’s are currently not available due to mechanical issues with the instrument. Figure 2: Observing the effects of eliminating a pair of disulfide bond on folding. A comparison was made between the folding abilities of LNRB original and mut 1,5 C  A_original. The samples in this figure were refolded overnight and overlaid with their reduced forms. The chromatogram shows a single thermodynamically preferred folded conformation for LNRB_original and three main folded conformations for mut 1,5 C  A_original. Figure 3: Observing the effects of eliminating the hydrophobic residue W52 on the folding of LNRB_original. A comparison was made between the folding abilities of LNRB original and mut W52  A52_ original. This chromatogram shows that although there was one preferred folded conformation reached for samples of the two constructs allowed to fold overnight, there were several additional conformations reached for the mut W52  A52_original construct. Figure 4: Comparison of the folding patterns for the four “original” constructs at 1.5 hours. The four different constructs of LNRB_original were refolded for 1.5 hours. The two constructs with only four cysteines (mut 1,5 C  A_original and mut 1,5 C  A mut W52  A52 original) both showed three major folded conformations after 1.5 hours whereas the wild type and the mut W52  A52_original showed one main folded conformation after 1.5 hours. However, the refolded mut W52  A52_original displayed additional conformations similar to the ones shown in Figure 3. Figure 6: Observing the effects of eliminating the hydrophobic residue W52 on the folding of LNRB_short. A comparison made between LNRB_short_0hr and mut W52  A52_short_0hr shows that the elimination of W52 did have some effects on the module. However, a comparison between LNRB_short_overnight and mut W52  A52_short_overnight shows that similar conformations were reached regardless of the W52 mutation. Original_overnight Mut 1,5 C  A Original_overnight Mut 1,5 C  A Original_reduced Original_reduced Mut W52  A52 Original_overnight Original_overnight Mut W52  A52 Original_reduced Original_reduced Mut W52  A52 Original Mut 1,5 C  A Mut W52  A52 Original Mut 1,5 C  A Original Original Figure 1: The 3-D structure of LNRB in relationship to LNRA and the α- helix of the heterodimerization (HD) domain. The light purple structure on the left is LNRB and the greenish - yellow structure on the right is LNRA. The α- helix of the HD domain is shown in the bottom center in red. W52, which is mutated to an alanine in some constructs, is shown in cyan blue. W42, which is located in the LNR-AB linker, is shown in navy blue. Located in LNRA is W29, which is shown in violet. The Ca 2+ ion is shown as a blue sphere. Figure 5 and Table 2: Distances between coordinating ligands and the Ca 2+. Measurements were made on PyMol and a criterion of < 3 Å was used to determine the Ca 2+ coordinating ligands in LNRB. The distances are listed in Table 2 on the right with their respective coordinating ligands. Electron Donors Residue Distance to Ca 2+ ( Å ) D722.68 D612.23 Y542.35 D572.65 H592.39 D752.44 --- Short _ 0hr ---Short_overnight ---mut W52  A52_Short_0hr ---mut W52  A52_Short_overnight