Try different scan-window size (2, 5, 10 & 15). What do you observe?
1. Calculate molecular weight and G+C content of the following DNA sequence tryseq. (BioEdit starts automatically)
(Edit>Select All sequences --- Sequence>Nucleic Acis>Nucleotide Composition)
DNA molecule: gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I
Length = 862 base pairs
Molecular Weight = 263023 Daltons, single stranded
Molecular Weight = 524606 Daltons, double stranded
G+C content = 53,83%
A+T content = 46,17%
Nucleotide Number Mol%
A 191 22,16
C 255 29,58
G 209 24,25
T 207 24,01
2. Translate into six frames. How many ORFs (Open Reading Frame) are predicted which are over 100 amino acids long
Copy the longest ORF into your word editor
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 1 to 732: Frame 1 244 aa
ATMISLVFVLLIGAAFATEDDKIVGGYECKAYSQTHQVSLNSGYHFCGGSLVNENWVVSAAHCYKSRVEVRLGEHNIKVTEGSEQFISSSRVIRHPNYSSYNIDNDIMLIKLSKPATLNTYVQPVALPTSCAPAGTMCTVSGWGNTMSSTADSNKLQCLNIPILSYSDCNNSYPGMITNAMFCAGYLEGGKDSCQGDSGGPVVCNGELQGVVSWGYGCAEPGNPGVYAKVCIFNDWLTSTMASY
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 264 to 1: Frame -2 88 aa
RDELLATFSHLDVVLAQTHLHTGLVAVSSRHNPVLIDQGATTEVVSRVQRHLVGLGVGLALIPSDDLVVLCGESGSNEQNEDQRNHGC
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 822 to 268: Frame -2 185 aa
SPVQKVDVDLERDVVELWDRAGGPKLGSDLVGGHGAGQPVIEDANLGVDTRVTGLSTSVTPGHNTLELTIAHHRATRVTLARVLASLQVSSTEHGVGDHARVGVVTVAVGQDGDVQALQLVAVSSGAHGVAPSRDGTHGASGGTAGGQSNRLHVGVEGGGFAQFDQHDVIIDVVGAVVGVTDHAG
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 529 to 356: Frame -1 58 aa
SCQGRSCYSRCRTGWGCSGTAACCCQQWSSWCCPIQRRYTWCQRGHSWWAEQQAARRC
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 530 to 709: Frame 2 60 aa
SPTPCSVLDTWREARTLARVTLVALWCAMVSSRVLCPGVTDVLSPVTLVSTPRFASSMTG
>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 697 to 533: Frame -1 55 aa
RCKPWRRHQGYRAQHIRNPRTQHPGAHHCTPQGHQSHPGKSPCLPPGIQHRTWRW
3. Close all BioEdit windows (except BioEdit) and create a new sequence file.(File>New Alignment --- Sequence>New sequence)
Paste the longest amino acid sequence into the "New Sequence" window and give the sequence a name (e.g. test1) and select a Sequence Type (Protein). Then Apply and close.
Calculate the molecular weight of the ORF and composition. Which two amino acids are most abundant?
Protein: test1
Length = 244 amino acids
Molecular Weight = 26129,17 Daltons
Amino Acid Number Mol%
Ala A 17 6,97
Cys C 12 4,92
Asp D 9 3,69
Glu E 10 4,10
Phe F 6 2,46
Gly G 24 9,84
His H 5 2,05
Ile I 13 5,33
Lys K 9 3,69
Leu L 16 6,56
Met M 7 2,87
Asn N 17 6,97
Pro P 10 4,10
Gln Q 7 2,87
Arg R 4 1,64
Ser S 27 11,07
Thr T 14 5,74
Val V 20 8,20
Trp W 4 1,64
Tyr Y 13 5,33
4. Try one or more programs predicting hydrophobic profiles. (e.g. Kyte-Doolittle) Which regions are most hydrophobic?
Try different scan-window size (2, 5, 10 & 15). What do you observe?
5. Select amino 225 -245 (Clic and drag). Run Helical Wheel Diagram (Sequence>Protein>Helical Wheel Diagram). Which anino acids are clustered together one one side of the helix?
6. To try to find out what protein this may be, we will use a BLAST search. (Accessory Application>BLAST>NCBI BLAST over the Internet). Past the amino acid sequence into the window and press Search. After a while you will se a web-page from NCBI (National Center for Biotechnology Information) with the sequences in the database that is most similar to our sequence.
Which sequence is most similar to our sequence? From which organism? Has the structure been solved? If yes, how many disulfide bridges are the in the structure?
LOCUS TRY1_SALSA 242 aa linear VRT 15-JUN-2002
DEFINITION Trypsin I precursor.
ACCESSION P35031
VERSION P35031 GI:464944
DBSOURCE swissprot: locus TRY1_SALSA, accession P35031;
class: standard.
created: Feb 1, 1994.
sequence updated: Feb 1, 1994.
annotation updated: Jun 15, 2002.
xrefs: gi: 64379, gi: 64380, gi: 64381, gi: 64382, gi: 64383, gi:
64384, gi: 422581, gi: 422582, gi: 422583, gi: 515295, gi: 809221
xrefs (non-sequence databases): MEROPSS01.151, InterProIPR001314,
InterProIPR001254, PfamPF00089, SMARTSM00020, PROSITEPS50240,
PROSITEPS00134, PROSITEPS00135
KEYWORDS Hydrolase; Serine protease; Digestion; Pancreas; Zymogen; Signal;
Multigene family; 3D-structure.
SOURCE Salmo salar (Atlantic salmon)
ORGANISM Salmo salar
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Actinopterygii; Neopterygii; Teleostei; Euteleostei;
Protacanthopterygii; Salmoniformes; Salmonidae; Salmo.
REFERENCE 1 (residues 1 to 242)
AUTHORS Male,R., Lorens,J.B., Smalas,A.O. and Torrissen,K.R.
TITLE Molecular cloning and characterization of anionic and cationic
variants of trypsin from Atlantic salmon
JOURNAL Eur. J. Biochem. 232 (2), 677-685 (1995)
MEDLINE 96035908
PUBMED 7556223
REMARK SEQUENCE FROM N.A.
TISSUE=Pancreas
REFERENCE 2 (residues 1 to 242)
UTHORS Smalas,A.O. and Hordvik,A.
TITLE Structure determination and refinement of benzamidine- inhibited trypsin from the North Atlantic salmon (Salmo salar) at 1.82-A resolution
JOURNAL Acta Crystallogr. D 49, 318-330 (1993)
REMARK X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS).
REFERENCE 3 (residues 1 to 242)
AUTHORS Smalas,A.O., Heimstad,E.S., Hordvik,A., Willassen,N.P. and Male,R.
TITLE Cold adaption of enzymes: structural comparison between salmon and
bovine trypsins
JOURNAL Proteins 20 (2), 149-166 (1994)
MEDLINE 95148588
PUBMED 7846025
REMARK X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS).
COMMENT -------------------------------------------------------------------
This SWISS-PROT entry is copyright. It is produced through a
collaboration between the Swiss Institute of Bioinformatics and
the EMBL outstation - the European Bioinformatics Institute.
The original entry is available from http://www.expasy.ch/sprot
and http://www.ebi.ac.uk/sprot
------------------------------------------------------------------.
[CATALYTIC ACTIVITY] Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.
[SUBCELLULAR LOCATION] Extracellular.
[SIMILARITY] BELONGS TO PEPTIDASE FAMILY S1.
FEATURES Location/Qualifiers
source 1..242
/organism="Salmo salar"
/db_xref="taxon:8030"
Protein 1..242
/product="Trypsin I precursor"
/EC_number="3.4.21.4"
Region 1..15
/region_name="Signal"
/note="POTENTIAL."
Region 16..20
/region_name="Propeptide"
/note="ACTIVATION PEPTIDE."
Region 21..242
/region_name="Mature chain"
/note="TRYPSIN I."
Region 22
/region_name="Beta-strand region"
Region 25..26
/region_name="Beta-strand region"
Bond bond(27,156)
/bond_type="disulfide"
Region 29..34
/region_name="Hydrogen bonded turn"
Region 33
/region_name="Variant"
/note="T -> A (IN TRYPSINS IA/IB)."
Region 35..39
/region_name="Beta-strand region"
Region 43..49
/region_name="Beta-strand region"
Bond bond(45,61)
/bond_type="disulfide"
Region 54..57
/region_name="Beta-strand region"
Region 59..61
/region_name="Helical region"
Site 60
/site_type="active"
/note="CHARGE RELAY SYSTEM."
Region 67..70
/region_name="Beta-strand region"
Region 74
/region_name="Beta-strand region"
Region 75..76
/region_name="Hydrogen bonded turn"
Region 83..92
/region_name="Beta-strand region"
Region 94..95
/region_name="Hydrogen bonded turn"
Region 97
/region_name="Beta-strand region"
Region 98..101
/region_name="Hydrogen bonded turn"
Region 102
/region_name="Beta-strand region"
Region 103
/region_name="Hydrogen bonded turn"
Site 104
/site_type="active"
/note="CHARGE RELAY SYSTEM."
Region 106..110
/region_name="Beta-strand region"
Region 124
/region_name="Beta-strand region"
Bond bond(129,229)
/bond_type="disulfide"
Region 132..133
/region_name="Hydrogen bonded turn"
Region 135..140
/region_name="Beta-strand region"
Bond bond(136,202)
/bond_type="disulfide"
Region 151..152
/region_name="Hydrogen bonded turn"
Region 153
/region_name="Beta-strand region"
Region 155..161
/region_name="Beta-strand region"
Region 164..170
/region_name="Helical region"
Bond bond(167,181)
/bond_type="disulfide"
Region 172..174
/region_name="Hydrogen bonded turn"
Region 177..178
/region_name="Hydrogen bonded turn"
Region 179..182
/region_name="Beta-strand region"
Region 185..186
/region_name="Hydrogen bonded turn"
Region 190
/region_name="Beta-strand region"
Site 190
/site_type="unclassified"
/note="REQUIRED FOR SPECIFICITY (BY SIMILARITY)."
Bond bond(192,216)
/bond_type="disulfide"
Region 193..194
/region_name="Hydrogen bonded turn"
Region 196..197
/region_name="Hydrogen bonded turn"
Site 196
/site_type="active"
/note="CHARGE RELAY SYSTEM."
Region 199..202
/region_name="Beta-strand region"
Region 203..204
/region_name="Hydrogen bonded turn"
Region 205..212
/region_name="Beta-strand region"
Region 219..220
/region_name="Hydrogen bonded turn"
Region 223..227
/region_name="Beta-strand region"
Region 228..230
/region_name="Helical region"
Region 232..240
/region_name="Helical region"
ORIGIN
1 mislvfvlli gaafateddk ivggyeckay sqthqvslns gyhfcggslv nenwvvsaah
61 cyksrvevrl gehnikvteg seqfisssrv irhpnyssyn idndimlikl skpatlntyv
121 qpvalptsca pagtmctvsg wgntmsstad snklqclnip ilsysdcnns ypgmitnamf
181 cagyleggkd scqgdsggpv vcngelqgvv swgygcaepg npgvyakvci fndwltstma
241 sy
//
7. Close all BioEdit windows and press the following file, which starts BioEdit again. You will se several sequences.
Select all sequences (Edit>Select All sequences). We will now try to align all these sequences (Accessory Application>ClustalW Multiple alignment). In the ClustalW Multiple Alignment window, clic for Full Multiple alignment, Calculate NJ tree and then press Run ClustalW.
After a while you will se a new window with the aligned sequences. There are three proteins that have a large deletion in the region around 450 to 550. Which? Vibcho, Vibsp and Vibalg
One sequence seems to have a deleted C-terminal. Who? ProK
8. To see which sequence are most related, we will run the phylogenetic program PROTPARS (Protein Sequence Parsimony Method) (Accessory Application>Protpars protein parsimony method). Which sequences are SerX most related to?
Protein parsimony algorithm, version 3.573c
One most parsimonious tree found:
+-----Vibcho
+--------5
! ! +--Vibsp
! +--6
+--4 +--Vibalg
! !
! ! +--Altsp
! ! +--3
--1 +--------2 +--AltspAS11
! !
! +-----SerX
!
+-----------------ProK
remember: this is an unrooted tree!