1. Identification and Characterization of Lin12/Notch Repeats (LNRs): Conclusion/Future Work: Our preliminary results indicate that although all LNRs, regardless of their origin, are small, acidic sequences, there are important subtle differences in the details of each LNR sequence that might shed light into their unique biological function within the larger multidomain protein scaffold. We have also found that some slots in the alignment of the LNRs predominantly contain a certain type of amino acid, either acidic, basic, hydrophobic, polar or aromatic. This compiled information, in the future, will be used in deciding which LNRs are relevant for further experimental characterization study and comparison of the bioinformatics data with experimental results will give us a clearer understanding of the characteristics of LNRs from such a diverse variety of protein families. References: 1.Notch Subunit Heterodimerization and Prevention of Ligand-Independent Proteolytic Activation Depend, Respectively, on a Novel Domain and the LNR Repeats. Cheryl lSanchez-Irizarry, Andrea C. Carpenter, Andrew P. Weng, Warren S. Pear, Jon C. Aster, and Stephen C. Blacklow. Molecular and Cellular Biology, Nov.2004, Vol.24, No.21. p 9265–9273. 2.The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine its Proteolytic Specificity. Henning B. Boldt, Kasper Kjaer-Sorensen, Michael T. Overgaard, Kathrin Weyer, Christine B. Poulsen, Lars Sottrup-Jensen, Cheryl A. Conovers, Linda C. Giudice, Claus Oxvig. Journal of Biological Chemistry, Sept. 2004, Vol. 279, No. 37, p. 38525-38531. 3.Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense. Peter Sperisen, Christoph D. Schmid, Philipp Bucher, Olav Zilian. PLoS Comput Biol. 1(6): e63. 2005. 4.“Number of Cysteines Histogram”. UCSC Genome Bioinformatics. Updated 12 Feb. 2004. Fig. 5: Physichochemical characteristics of LNRs. Each LNR sequence is characterized using ExPASy Proteomics. Information such as the theoretical pI and total number of residues tells us that all LNRs are acidic and are less than 45 amino acids long. This tables shows a few of the characteristics and compiled information for some selected LNR sequences. Fig. 1: Domain organization of different classes of proteins that contain LNRs. In the Notch protein the LNRs (represented as yellow ovals) are found in a tandem block of three, while in the PAPP the first two tandem LNRs are separated from the third LNR by ~1000 amino acids. Human Stealth Protein contains two LNRs while the Fly Stealth protein contains only a single LNR Human Stealth: Fly Stealth: CR1 CR2CR4CR3 LNRALNR B CR4CR3 CR1CR2 LNRA Proteolytic Domain 45321 NEC LNRs RAM Ankyrin repeats Transactivatio n Domain Transmembrane region EGF-Like repeats Notch: PAPP: Fig. 2: Websites and Tools NameAccessionSequenceResidues# CysMWpI (Theor.)# neg.# Pos. Instability Index # aliphatic# Aromatic# Basic# AcidicTotal #Ex. Co. (all half) hN1 LNRAP46531EEACELPECQEDAGNKVCSLQCNNHACGWDGGDCS1447-148163715.93.898192.14101213355875 hN1 LNRBP46531 LNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDG FDCQ1482-152364827.24.285253.47773134212865 hN1 LNRCP46531 RAEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCA1524-156364484.74.129220.6394414408855 hN2 LNRAQ04721PATCLSQYCADKARDGVCDEACNSHACQWDGGDC1422-145563593.84.176252.4110239347365 hN2 LNRBQ04721 LTMENPWANCSSPLPCWDYINNQCDELCNTVECLFDNFE CQ1457-149764804.33.267050.62750154112865 hN2 LNRCQ04721 GNSKTCKYDKYCADHFKDNHCDQGCNSEECGWDGLDC A1498-153564261.54.648436.1174612388855 hN3 LNRAQ9UM47EPRCPRAACQAKRGDQRCDRECNSPGCGWDGGDCS1384-141863785.16.316669.218169355875 hN3 LNRBQ9UM47 LSVGDPWRQCEALQCWRLFNNSRCDPACSSPACLYDNFD CH1419-145964722.24.755395.14954104112865 hN3 LNRCQ9UM47 AGGRERTCNPVYEKYCADHFADGRCDQGCNTEECGWD GLDCA1460-150164616.94.359434.811245 428855 hN4 LNRAQ5STG5CEGRSGDGACDAGCSGPGGNWDGGDCS1180-120642490.53.715148.3211116275750 Q5STG5PGAKGCEGRSGDGACDAGCSGPGGNWDGGDCS1175-120642900.94.045237.0314126325750 hN4 LNRBQ5STG5 LGVPDPWKGCPSHSRCWLLFRDGQCHPQCDSEECLFDG YDCE1207-124864830.34.578383.37955104212865 hN4 LNRCQ5STG5 TPPACTPAYDQYCHDHFHNGHCEKGCNTAECGWDGGDC R1249-128764297.65.26269.488469398855 mN1 LNRAQ01705EEACELPECQVDAGNKVCNLQCNNHACGWDGGDCS1446-1480637133.957174.68111213355875 mN1 LNRBQ01705 LNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDG FDCQ1481-152264827.24.285253.47773134212865 mN1 LNRCQ01705 LTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLD CA1523-156264471.73.939125.4594314408855 dN LNRAP07207RAMCDKRGCTECQGNGICDSDCNTYACNFDGNDCS1479-1513737714.176360.4672311351865 Slot #TotalHigh. Perc. # Hydr. (G, A, V, L, I, M, P) # Arom. (F, Y, W) # Basic (H, K, R) # Acidic (D, E) # Polar (S, C, T, N, Q)% 1978% L9100% Hydrophobic 21040% N31660% Polar 31233% F8467% Hydrophobic 41225% N512442% Hydrophobic 51833% D4146333% Acidic 61947% P132468% Hydrophobic 72931% E78210234% Acidic 82921% K1583352% Hydrophobic 92928% N7141759% Polar 1029100% C29100% Polar 11250% V, E1150% Acid/Hydr. 12333% D, V, T11133% Hydr/Acid/Polar 13333% Y, S, L11133% Hydr/Arom/Polar 143 33% Q, N, R1267% Polar 151050% N111770% Polar 161060% P71270% Hydrophobic 171833% L111661% Hydrophobic 182825% Y5817729% Aromatic 193222% D1348741% Hydrophobic 203222% Q1373941% Hydrophobic Fig. 4: Alignment Slots Statisticss –Some of Them. Each slot (see Fig. 3) is analyzed for the most abundant amino acid (Column 3 – Highest Percentage) and then analyzed for different types of amino acids (Columns 4- 8). Many slots are made predominantly of a certain type of amino acid. Information for slots 1-20 is shown. LNR CLNR ALNR B LNR A LNR B LNR C Fig. 3: LNR alignment. All LNRs are aligned based on the position of key structural amino acids such as the cysteines and the aspartic acids (highlighted in red and green, respectively). Each “slot” is numbered (on top). This alignment allows us to see similarities and trends in each “slot”, giving us further clues to LNR characteristics. LNRA LNRB LNRC Human Notch1 LNRA LNRB LNRC Mouse Notch1 LNRA LNRB LNRC Human Notch4 LNRA LNRB LNRC Human Notch2 LNRA LNRB LNRC Human Notch3 LNRA LNRB LNRC Fruit Fly Notch LNRA LNRB LNRC Frog Notch LNRA LNRB LNRC Zebra Fish Notch Green Algae Gluc TraB Gluc TraA Stealth PAPP A PAPP A2 PAPP E Nematode Notch 15101520253035404550 Slot # A Bioinformatics Approach Fathima F. Jahufar, Framingham High School ’07. Didem Vardar-Ulu, Chemistry Department Acknowledgements - National Science Foundation Research Experiences for Undergraduates (NSF- REU) in Chemistry and Physics - Professor Didem Vardar-Ulu, Christina Hao, Sharline Madera, and Ursela Siddiqui. Abstract Lin12 Notch Repeats (LNRs) are Ca 2+ binding, cysteine-rich protein domains. They were first found in a block of three in a transmembrane receptor protein called Notch. Since then they have also been found in other types of multidomain proteins such as the Pregnancy-associated Plasma Protein (PAPP) and Stealth proteins. In these proteins, the LNRs are present in a variety of different numbers and arrangements. For this project, we have used a variety of different bioinformatics tools to identify, align, and compile information on different LNRs from different protein sources. These tools include BLAST, ClustalW, ExPASy Proteomics Tools, and UniProt. Using these tools, we have been able to compile a list of different LNRs along with certain physicochemical properties of each, including the theoretical pI, the molecular weight, the number of acidic and basic residues and the extinction coefficients. We have also broken down the percentages of each amino acid and each type of amino acid in each residue position relative to the cysteines. Our preliminary results indicate that although all LNRs, regardless of their origin, are small, acidic sequences. There are important subtle differences in the details of each LNR sequence that might shed light into their unique biological function within the larger multidomain protein scaffold. The compilations presented in this work are useful in comparing different LNRs and deciding which LNRs would be valuable for further studies.. Introduction: Lin12 Notch Repeats (LNRs) are relatively short protein domains (only about 35-40 amino acids long) found in a variety of different protein families. LNRs were first found in a block of three in Notch protein, a transmembrane receptor protein. In this protein, LNRs help maintain the receptor in a resting, metalloprotease-resistant conformation prior to ligand binding (1). LNRs are also found in other multidomain proteins such as PAPP proteins and Stealth proteins. PAPP proteins, like the Notch, have three LNRs. However, the third LNR is separated from the second LNR by more than 1000 amino acids (2). LNRs in PAPP are thought to determine the proteolytic specificity of PAPP, which cleaves insulin-like growth factor-binding proteins (2). In Stealth, LNRs come in ones or twos, but are not found in all Stealth proteins (3). Average natural abundance of cysteine in proteins is about 2.3% (4). However, most LNRs are ~ 15-17% cysteine. Hence, they are very cysteine rich and require Ca 2+ to fold properly into their native forms. Most LNRs have six cysteines, while a few have only four. These cysteines help to form three (or two) specific disulfide bridges that help give LNRs their structure. LNRs also contain several aspartic acids and asparagines that coordinate the binding of Ca 2+ ions. Using bioinformatics to study LNRs involves the use of websites such as UniProt, BLAST, ClustalW2, and ExPASy Proteomics Tools. UniProt allows keyword/ text searches to identify amino acid sequences from different data bases. It also matches input sequences to sequences within proteins in a database and provides basic information about these proteins. Protein BLAST (Basic Local Alignment Search Tool) compares amino acids sequence inputs to those in the protein database and outputs significant matches. ClustalW2 is an online tool that aligns multiple amino acid sequences facilitating one to one amino acid comparisons. Finally, ExPASy (Expert Protein Analysis System) Proteomics tools allow information to be gathered and predictions to be made about amino acids sequences. We have used UniProt and BLAST to first identify different LNR sequences within the protein database and to determine their location within their corresponding protein sources. Then, we used ClustalW to align these LNRs, after which we improved these automated alignments manually based on the position of the cyteines and the Ca 2+ coordinating residues that define an LNR. Finally, in order to better understand and predict the biochemical and biophysical characteristics of LNRs, we used EXPASY Proteomics Tools to compile a list of physicochemical properties for each of the identified LNR sequences. The alignments of the LNRs (each slot numbered) and small sections of the tables detailing the properties of the LNRs and of each slot in the alignments are presented here.