Temwachi Mphande

Professor Borgon

BSC 3403C

12/02/17

BLAST

1. A BLAST search allows you to compare a particular sequence of DNA/RNA to other sequences in the
GenBank Database and this allows for even the Protein and DNA databases of several different species to
be accessed.

2. Western Lowland Gorilla (100%), Rhesus Macaque (98%), and Peter’s Angolan Colobus (98%)

NCBI Gene
3. Vinculin is a cytoskeletal protein associated with cell-to-cell and cell-matrix junctions. It is thought to
function as one of several interacting proteins involved in anchoring F-actin to the membrane

4. 10q22.2

5. LOC107984244

6. L954del, R975W, and A934V. They cause Sporadic/Familial Dilated Cardiomyopathy

7. HIV and Vinculin Interactions: HIV-1 Nef co-localizes with vinvulin inside podosomes in human
monocyte-derived macrophages. HIV-1 Nef induces polarization of vinculin, a molecule important for
dendritic cell (DC) adhesion, motility, and maturation, at one pole of DCs corresponding to the substrate-
adhering portion or leading edge of the cell. HIV-1 MA co-localizes with beta2 integrin, alphaM and
alphaX integrins in the intracellular thick electron-dense membrane compartments, which contain talin,
vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton.

8. Ponsin, Actin, and α-Catenin

9. α-Catenin and β-Catenin

10. It is found on the same chromosome (Chromosome 10) in Chimpanzees.

NCBI GenBank
11. Coding Sequence

12. 22 Exons

13. 10 Phosphorylation Sites. These are for the gene function regulation.

PubMed
14. Turner CE, Complete sequence of human vinculin and assignment of the gene to chromosome 10,
1990. This sequence was determined before the publishing of the human genome project.

15. Phosphorylation

16. 𝑐𝐷𝑁𝐴 Library

17. Sanger Sequencing

18. No they don’t. This is because different nucleotides can code for the same protein.

19. Southern Blotting and Agarose Gel Electrophoresis

20. There is at least one additional isoform of vinculin, termed metavinculin, which has a higher apparent
molecular weight than vinculin and is expressed specifically in muscle

ExPASy
21. 94. It is not at position 1 because the 5’ UTR (untranslated region) lies upstream of the start codon
ATG. This is necessary for the regulation of translation. The 5’ UTR can be up to 100 nucleotides long in
Eukaryotes.

22. Adenine. The Adenines form the Poly A Tail of the 𝑚𝑅𝑁𝐴 and this is necessary for translation,
𝑚𝑅𝑁𝐴 stability, and nuclear export.

23. 5’3’ Frame 2. PVHFT are the first 5 amino acids after the Met.

24.

ID VIRT8316 Unreviewed; 1066 AA.

AC VIRT8316;
DE Translation of nucleotide sequence generated on ExPASy
DE on 02-Dec-2017 by 75.102.129.45.
CC -!- This virtual protein sequence will automatically be deleted
CC from the server after a few days.
DR SWISS-2DPAGE; VIRT8316; VIRTUAL.
SQ SEQUENCE 1066 AA; C9B67AA072009EBD CRC64.
MPVFHTRTIE SILEPVAQQI SHLVIMHEEG EVDGKAIPDL TAPVAAVQAA VSNLVRVGKE
TVQTTEDQIL KRDMPPAFIK VENACTKLVQ AAQMLQSDPY SVPARDYLID GSRGILSGTS
DLLLTFDEAE VRKIIRVCKG ILEYLTVAEV VETMEDLVTY TKNLGPGMTK MAKMIDERQQ
ELTHQEHRVM LVNSMNTVKE LLPVLISAMK IFVTTKNSKN QGIEEALKNR NFTVEKMSAE
INEIIRVLQL TSWDEDAWAS KDTEAMKRAL ASIDSKLNQA KGWLRDPSAS PGDAGEQAIR
QILDEAGKVG ELCAGKERRE ILGTCKMLGQ MTDQVADLRA RGQGSSPVAM QKAQQVSQGL
DVLTAKVENA ARKLEAMTNS KQSIAKKIDA AQNWLADPNG GPEGEEQIRG ALAEARKIAE
LCDDPKERDD ILRSLGEISA LTSKLADLRR QGKGDSPEAR ALAKQVATAL QNLQTKTNRA
VANSRPAKAA VHLEGKIEQA QRWIDNPTVD DRGVGQAAIR GLVAEGHRLA NVMMGPYRQD
LLAKCDRVDQ LTAQLADLAA RGEGESPQAR ALASQLQDSL KDLKARMQEA MTQEVSDVFS
DTTTPIKLLA VAATAPPDAP NREEVFDERA ANFENHSGKL GATAEKAAAV GTANKSTVEG
IQASVKTARE LTPQVVSAAR ILLRNPGNQA AYEHFETMKN QWIDNVEKMT GLVDEAIDTK
SLLDASEEAI KKDLDKCKVA MANIQPQMLV AGATSIARRA NRILLVAKRE VENSEDPKFR
EAVKAASDEL SKTISPMVMD AKAVAGNISD PGLQKSFLDS GYRILGAVAK VREAFQPQEP
DFPPPPPDLE QLRLTDELAP PKPPLPEGEV PPPRPPPPEE KDEEFPEQKA GEVINQPMMM
AARQLHDEAR KWSSKGNDII AAAKRMALLM AEMSRLVRGG SGTKRALIQC AKDIAKASDE
 VTRLAKEVAK QCTDKRIRTN LLQVCERIPT ISTQLKILST VKATMLGRTN ISDEESEQAT
EMLVHNAQNL MQSVKETVRE AEAASIKIRT DAGFTLRWVR KTPWYQ
//
Sequence in FASTA format
25. 1066 amino acids. With Ala being the most common at 12.5%

26.

27. Cation Exchange Chromatography with a buffer of 𝑝𝐻 4.83 or lower. Use negatively charged beads
for protein binding.

28. 𝐴 = 𝜀𝑏𝑐

Sequence Alignment
29.

10 20 30 40 50 60
| | | | | |
Chicken MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVSAVQAAVSNLVRVGKE
Turkey MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVSAVQAAVSNLVRVGKE
Lizard MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Humanx0 MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Pigxxx5 MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Mousex1 MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Cowxxx4 MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Frogxx10 MPVFHTKTIESILEPVAQQISHLVIMHEEGEVDGKAIPELTAPVAAVQAAVSNLVRVGKE
Seaxxx11 MPVFHTKTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE
Zebrafish MPVFHTKTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTSPVAAVQAAVSNLVRVGKE
Mosquito MPVFHTKTIESILEPVAQQVSRLVILHEEAE-DGNAMPDLSRPVQAVSLAVTNLVRVGRE
Fruitx6 MPVFHTKTIESILDPVAQQVSRLVILHEEAE-DGNAMPDLSRPVQVVSAAVANLVKVGRD
Roundworm MPVFHTKTIENILEPVAQQVSRLVILHEEAN-DGNAMPDLTGPVGMVSRAVGNLIQVGYD
******:***.**:*****:*:***:***.: **:*:*:*: ** *. ** **::** :
Prim.cons. MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKE

70 80 90 100 110 120

| | | | | |
Chicken TVQTTEDQILKRDMPPAFIKVENACTKLVRAAQMLQADPYSVPARDYLIDGSRGILSGTS
Turkey TVQTTEDQILKRAMPPAFIKVENACTKLVRAAQMLQADPYSVPARDYLIDGSRGILSGTS
Lizard TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQTDPYSVPARDYLIDGSRGILSGTS
Humanx0 TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTS
Pigxxx5 TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTS
Mousex1 TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTS
Cowxxx4 TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTS
Frogxx10 TVQTTEDQIMKRDMPPAFIKVENACAKLVQAAQMLHADPYSVPARDYLIDGSRGILSGTS
Seaxxx11 TVQTTEDQIMKRDMQPAFIKVENACTKLVQAASMLKADPYSVPARDYLIDGSRGILSGTS
Zebrafish TVQTTEDKIMKRDMPSAFIKVENACAKLVEAAQMLRTDPYSVPARDYLIDGSRGILSGTS
Mosquito TIHNSDDDILKQDMPSALARVENAAQLLEEASGMLKMDPFSGPARKKLIEGSRGILQGTS
Fruitx6 TINSSDDKILRQDMPSALHRVEGASQLLEEASDIVRSDPYSGPARKKLIEGSRGILQGTS
Roundworm TCDHSDDRILQQDMPPALQRVEGSSKLLEESSYSLKHDPYSVPARKKLIDGARGILQGTS
* . ::* *::: * .*: :**.:. * .:: :: **:* ***. **:*:****.***
Prim.cons. TVQTTEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTS

30. The most similarities are seen in the beginning of the sequence. The identical residues are more than
the different residues hence the similarities in the different species. The overall identity is 25.65% .
However, it needs to be more than 30% for the species to be considered identical. Some of the species
have different number of residues from the other species. Such as the human with 1066 and the
Roundworm with 1010. There are insertion towards the end of the sequence in some of the species. This
shows that some of these species originated from a common origin, but because of evolution have formed
differences.

Phylogenetic Tree
31. The Phylogenic Tree shows that the Roundworm has the least similarity with the other animals, but it
does have common links with them. The Mosquito and the Fruit-fly have similar genes. The Zebrafish
has its own origin. So does the Seabass, Frog, and Lizard. The closer down the tree, the more similarities
the species have. Therefore, the Turkey and Chicken have the same origin and are very similar genes to
each other. The Pig and Human have similar genes as well. The mouse and the cow have similar genes.
However, the tree shows that at some point the origin is the same, but differences occur that lead to the
branching out of the tree. This confirms that all genes are similar at some point.
Secondary Structure Prediction
32. α-helix is 66.23%, β-sheet is 4.13%, and random coil is 29.55%.

33.

Hierarchical Neural Network result for :

UNK_591150
Abstract Guermeur, Y. PhD Thesis
View HNN in: [AnTheProt (PC) , Download...] [HELP]

10 20 30 40 50 60 70
| | | | | | |
MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKETVQTTEDQIL
ccccccchhhhhhhhhhhhcceeeeeeccccccccccchhhhhhhhhhhhhhhhhhhcccchhchhhhhh
KRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTSDLLLTFDEAEVRKIIRVCKG
hhccccceeeeechhhhhhhhhhhhhccccccccchhehccccceeccchhhhehhchhhhhhhhhhhhh
ILEYLTVAEVVETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVMLVNSMNTVKELLPVLISAMK
hhhhhhhhhhhhhhhhhhhhhhcccchhhhhhhhhhhhhhhhhhhhheeeehcccchhhhhhhhhhhhhe
IFVTTKNSKNQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDEDAWASKDTEAMKRALASIDSKLNQA
eeeeecccccchhhhhhhccccchhhhhhhhhhhhhhheeccccccccccchhhhhhhhhhhhhhhhhhh
KGWLRDPSASPGDAGEQAIRQILDEAGKVGELCAGKERREILGTCKMLGQMTDQVADLRARGQGSSPVAM
hhccccccccccchhhhhhhhhhhhhcccccehccchhhhhcccchhhhhhhhhhhhhhhccccccchhh
QKAQQVSQGLDVLTAKVENAARKLEAMTNSKQSIAKKIDAAQNWLADPNGGPEGEEQIRGALAEARKIAE
hhhhhhhcchhhhhhhhhhhhhhhhhhccchhhhhhhhhhhhhhhcccccccchhhhhhhhhhhhhhhhh
LCDDPKERDDILRSLGEISALTSKLADLRRQGKGDSPEARALAKQVATALQNLQTKTNRAVANSRPAKAA
hhcccccchhhhhhhhhhhhhhhhhhhhhhhcccccchhhhhhhhhhhhhhhhhhhcccccccccchhhh
VHLEGKIEQAQRWIDNPTVDDRGVGQAAIRGLVAEGHRLANVMMGPYRQDLLAKCDRVDQLTAQLADLAA
hhhcchhhhhhhhhcccccccccchhhhhhhhhhhhhhhhhhccccchhhhhhhhcchhhhhhhhhhhhh
RGEGESPQARALASQLQDSLKDLKARMQEAMTQEVSDVFSDTTTPIKLLAVAATAPPDAPNREEVFDERA
hccccchhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhccchhhheeehhhhccccccccccchhhhhh
ANFENHSGKLGATAEKAAAVGTANKSTVEGIQASVKTARELTPQVVSAARILLRNPGNQAAYEHFETMKN
hhhhhcccccchhhhhhhhhccccccchhhhhhhhhhhhhhcchhhhhhhhhhhccccchhhhhhhhhhh
QWIDNVEKMTGLVDEAIDTKSLLDASEEAIKKDLDKCKVAMANIQPQMLVAGATSIARRANRILLVAKRE
hhhhhhhhhhhhhhhhccccchhhhhhhhhhhhhhhhhhhhhhcccchhhhhhhhhhhhhhhhhhhhhhh
VENSEDPKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLDSGYRILGAVAKVREAFQPQEP
hccccccchhhhhhhhhhhhhhhhhhhhhhhhhhhccccccccchchhhhhhhhhhhhhhhhhhhccccc
DFPPPPPDLEQLRLTDELAPPKPPLPEGEVPPPRPPPPEEKDEEFPEQKAGEVINQPMMMAARQLHDEAR
ccccccccccceeeccccccccccccccccccccccccccccccccccccchhhhhhhhhhhhhhhhhhh
KWSSKGNDIIAAAKRMALLMAEMSRLVRGGSGTKRALIQCAKDIAKASDEVTRLAKEVAKQCTDKRIRTN
hhhhcchhhhhhhhhhhhhhhhhhhhhccccchhhhhhhhhhhhhhhhhhhhhhhhhhhhhhcchhhhhh
LLQVCERIPTISTQLKILSTVKATMLGRTNISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRT
hhhhhhcccccchhhhhhhhhhhhhccccccccchhhhhhhhhhhhhhhhhhhhhhhhhhhhhhheheec
DAGFTLRWVRKTPWYQ
ccceeeeeeecccccc
Sequence length : 1066
HNN :
Alpha helix (Hh) : 707 is 66.32%
310 helix (Gg) : 0 is 0.00%
Pi helix (Ii) : 0 is 0.00%
Beta bridge (Bb) : 0 is 0.00%
Extended strand (Ee) : 44 is 4.13%
Beta turn (Tt) : 0 is 0.00%
Bend region (Ss) : 0 is 0.00%
Random coil (Cc) : 315 is 29.55%
Ambiguous states (?) : 0 is 0.00%
Other states : 0 is 0.00%
Prediction result file (text): [HNN]

User : public@75.102.129.45. Last modification time : Sat Dec 2 21:38:52 2017. Current time
: Sat Dec 2 21:38:52 2017

RCSB Protein Data Bank – 3D Structure

34. Cell Adhesion

35. 4.7% Ramachandran Outliers. This means that there are residues in which the combination of φ and
ψ torsion angles are unusual.

36. MSE. They are modified because they are nucleotide derivatives of the standard amino
acids/nucleotides.

37. Resolution is 2.9 Å. The space group is P 1 21 1. High-resolution structures, with resolution
values of 1 Å or so, are highly ordered and it is easy to see every atom in the electron density map.
A space group is the symmetry group of a configuration in space and allows one to visualize the
symmetry of the protein structure being analyzed.

38. The structure is mostly made up of α-helices. There are some β-sheets towards the ends of the strand,
but not many. There is some random coiling as well, but not a lot of this. The secondary protein
prediction was accurate.