BIO60404:

Eukaryotic Cell Biology

Practical 6
Computation Analysis Methods in Molecular Cell Biology

Name Student ID

Ang Jing Jie 0330393

Annur Najaa bt Zainal Abidin 0325632

G. U. Chobna d/o Geneshwaren 0328532

Marliah binti Abdul Ghanie 0331520

Nur Syafiqah bt Husain 0333589

Ramkhalawon Dikshita 0332052

Lecturer: Dr. Tang

Submission date: 24th June 2018

Analysis
Protein Sequence 1 (in Fasta Format)
MDRRMWGAHVFCVLSPLPTVLGHMHPECDFITQLREDESACLQAAEEMPNTTLGCPATWDGLLCWPTAGS
GEWVTLPCPDFFSHFSSESGAVKRDCTITGWSEPFPPYPVACPVPLELLAEEESYFSTVKIIYTVGHSIS
IVALFVAITILVALRRLHCPRNYVHTQLFTTFILKAGAVFLKDAALFHSDDTDHCSFSTVLCKVSVAASH
FATMTNFSWLLAEAVYLNCLLASTSPSSRRAFWWLVLAGWGLPVLFTGTWVSCKLAFEDIACWDLDDTSP
YWWIIKGPIVLSVGVNFGLFLNIIRILVRKLEPAQGSLHTQSQYWRLSKSTLFLIPLFGIHYIIFNFLPD
NAGLGIRLPLELGLGSFQGFIVAILYCFLNQEVRTEISRKWHGHDPELLPAWRTRAKWTTPSRSAAKVLT
SMC

BLASTp
>NP_000814.2 growth hormone-releasing hormone receptor precursor [Homo sapiens]

tBLASTn
>NM_000823.3 Homo sapiens growth hormone releasing hormone receptor (GHRHR), mRNA

Diagram 1: Origin nucleotide sequence for Protein Sequence 1, growth hormone-releasing

hormone receptor precursor of Homo sapiens.
 Figure 2 : The molecular weight and the pI (isoelectric point) of the receptor for protein
sequence 1.

The above information was obtained by running the Protein Sequence 1 using EMBOSS
Pepstats. It was determined that the sequence consists of 423 amino acids with molecular weight
of protein receptor of 47402.08 daltons while the pI value (isoelectric point) of the protein
receptor is 6.7114.
Figure 3: Properties of residues determined by the Physico-chemical properties of the receptor
of Protein Sequence 1
 Figure 4: Kyte-Doolitle Hydropathy plot of Protein Sequence 1

Figure 4 was obtained by running Protein Sequence 1 using EMBOSS PepWindow. The window
size used was 19. Cut-off point was determined to be at 1.6 on the y-axis and a total number of 7
peaks were observed at y-axis >1.6. Since there are 7 domains transmembrane protein, it can be
concluded that the receptor protein has the characteristics of G-protein coupled receptor (GPCR).

Protein Sequence 2 (FASTA format)

MGLMPLTKEVAKGSIGRGVLPAVELAIEQIRNESLLRPYFLDLRLYDTECDNAKGLKAFYDAIKYGPNHL
MVFGGVCPSVTSIIAESLQGWNLVQLSFAATTPVLADKKKYPYFFRTVPSDNAVNPAILKLLKHYQWRRV
GTLTQDVQRFSEVRNDLTGVLYGEDIEISDTESFSNDPCTSVKKLKGNDVRIILGQFDQNMAAKVFCCAY
EENMYGSKYQWIIPGWYEPSWWEQVHAEANSSRCLRKHLLTAMEGYIGVDFEPLSSKQIKTISGKTPQQY
EREYNNKRSGVGPSKFHGYAYDGIWVIAKTLQRAMETLHASSRHQRIQDFNYTDHTLGKIILNAMNETNF
FGVTGQVVFRNGERMGTIKFTQFQDSREVKVGEYNAVADTLEIINDTIRFQGSEPPKDKTIILEQLRKIS
LPLYSILSALTILGMIMASAFLFFNIKNRNQKLIKMSSPYMNNLIILGGMLSYASIFLFGLDGSFVSEKT
FETLCTVRTWILTVGYTTAFGAMFAKTWRVHAIFKNVKMKKKIIKDQKLLVIVGGMLLIDLCILICWQAV
DPLRRTVERYSMEPDPAGRDISIRPLLEHCENTHMTIWLGIVYAYKGLLMLFGCFLAWETRNVSIPALND
SKYIGMSVYNVGIMCIIGAAVSFLTRDQPNVQFCIVALVIIFCSTITLCLVFVPKLITLRTNPDAATQNR
RFQFTQNQKKEDSKTSTSVTSVNQASTSRLEGLQSENHRLRMKITELDKDLEEVTMQLQDTPEKTTYIKQ
NHYQELNDILNLGNFTESTDGGKAILKNHLDQNPQLQWNTTEPSRTCKDPIEDINSPEHIQRRLSLQLPI
LHHAYLPSIGGVDASCVSPCQPHRQPPSQTCATLFPSYGLGPVRVGGLGPGPPPGQNHTEQKQLLQEHCQ
LWLWRSWRHGWPCGTAQMARGTQDGGTWHLTSRLICEVFILSQRRGMEMTSSFMSAKEEELGYLKLCKKK
KKKS

BLASTp
>XP_011975863.2 gamma-aminobutyric acid type B receptor subunit 2

tBLASTn
>XM_003995690.5 PREDICTED: Felis catus gamma-aminobutyric acid type B receptor subunit
2 (GABBR2)
Diagram 5: Origin nucleotide sequence for Protein Sequence 2, Felis catus gamma-
aminobutyric acid type B receptor subunit 2 (GABBR2)
 Figure 6 : The molecular weight and the pI (isoelectric point) of the receptor for protein
sequence 2.

The above information was obtained by running the Protein Sequence 2 using EMBOSS
Pepstats. It was determined that the sequence consists of 984 amino acid with molecular weight
of the protein receptor 111483.66 daltons while the pI value (isoelectric point) of the protein
receptor is 8.7198 .
Figure 7: Properties of residues determined by the Physico-chemical properties of the receptor
of Protein Sequence 2.
 Figure 8: Kyte-Doolitle Hydropathy plot of Protein Sequence 2.

Figure 8 was obtained by running Protein Sequence 2 using EMBOSS PepWindow. The window
size used was 19. Cut-off point was determined to be at 1.6 on the y-axis and a total number of 7
peaks were observed at y-axis >1.6. Since there are 7 domains transmembrane protein, it can be
concluded that the receptor protein has the characteristics of gamma-aminobutyric acid type B
receptor subunit 2.

Multiple Sequence Alignment

 Figure 9: Alignment sequence of nucleotide sequence 1 and 2

Figure 9 shows the similarity of both nucleotide sequence 1 and 2. When compared, nucleotide
sequence 1 and 2 gives off a similarity percentage of 20.5%.

Procedure
The computational analysis is used to determine the unknown type of sequence, origin of
species, and the likely function of the sequence.The BLAST Database searching used to obtained
the similarity between query sequence and sequences in a nucleotide database (using BLASTN)
or peptide database (using BLASTP). Multiple sequence alignment is extremely computationally
intensive. Huge numbers were involved in evaluating all possible alignments between 2
sequences allowing for gaps. ClustalW has been ensured to be easily used and accurate because
of the under continuing development. Progressive alignment was used by Clustal as a shortcut.
The most closely related sequences were aligned first. Each sequence was then added according
to similarity until all sequences were aligned. Brief description on the functions of PepInfo,
PepWindow and PepStats were known. Protein sequence Homo sapiens melanocortin 4 receptor
was used. Protein characteristics were then recorded before the number of membrane-spanning
domains for MC4R were predicted from Kyte-Doolittle plot.

Flow cytometers utilized the lasers at specific wavelengths to measure cells labeled with
fluorescent antibodies (Flow Metric 2018). The electronics system in the detectors converted the
light signals into electronic signals then processed and stored in a computer (Sally Robertson
2014). The computer software analyzed and the data is represented to show different cell
parameters including size, shape, granularity and frequency of antibody–labeled cells (Flow
Metric 2018).

Immunofluorescence used specific antibodies which have been chemically conjugated to

fluorescent dyes. These labeled antibodies bind directly or indirectly to cellular antigens.
Biological applications such that evaluation of cells in suspension, cultured cells, tissue, beads
and in microarrays are used in the technique (Svar Life Science 2018).

Justification
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE is a common method that is used to determine the molecular weight of proteins. The
proteins are loaded in a discontinuous polyacrylamide gel and the loading buffer that contains
sodium dodecyl sulfate (SDS) will denature the protein during the electrophoresis. SDS is an
anionic detergent which the negative charges are able to disrupt the molecular conformation
within a wide pH range thereby destroyed the characteristic properties of protein and are strongly
attracted towards the positive end of the gel, including membrane proteins. The smaller charged
molecules move through the polyacrylamide gel matrix with a faster rate as compared to the
large molecules will restrain. This is due to the SDS-denatured polypeptides have about the same
charge-to-mass ratio and the final separation is rely on the differences in relative molecular mass
of polypeptides. The major difference between agarose gel and polyacrylamide gel is that the
agarose gel do not have the uniform pores and only allows the proteins larger than 200kDa to
pass though. However, polyacrylamide gels have a small range of separation but high resolving
power. Besides that, isoelectric focusing is the one of the technique that involved in the Commented [1]: marlia, should i include this
sentence?
separation without the effect of molecular charge (Caprette 2012).
Commented [2]: yeszz

This setup can be used to identify the growth hormone releasing hormone receptor
(GHRHR) for the Homo sapiens and the gamma-aminobutyric acid type B receptor subunit 2
(GABBR2) for the Felis catus. The specific molecular weights of both receptors which able to
obtain different results by running SDS-PAGE. There is only 20.5% of the similarity between
this two protein sequences, hence the membrane proteins can be identified through the SDS-
PAGE. To determine the unknown receptors, growth hormone releasing hormone receptor
(GHRHR) for the Homo sapiens and the gamma-aminobutyric acid type B receptor subunit 2
(GABBR2) for the Felis catus should be carried out with positive and negative controls. The
final outcome of the gel electrophoresis under positive control is able to calculate the molecular
weight of bands with the ladder acts as a guide.

Western blot
Western blotting is an important analytical technique used in cell and molecular biology. With
the help of western blot, researchers are able to identify specific proteins from a complex mixture
of proteins extracted from different type of cells. It is also a lab technique which is used to
detect proteins in a tissue or blood sample and helps researchers identify specific protein
molecules in a complex mixture of proteins. This technique is also known as an immunoblot
since antibodies are used to mark target protein. This technique can be achieved by three ways
which are: separation by size, transfer to a solid support, and marking target protein using a
proper primary and secondary antibody to visualize. Furthermore, it is an effective technique for
studying protein expression in the lab. By stripping the antibody after immunodetection,
identification of even multiple protein targets is possible using this technique. Western blot is
used in research to separate and identify proteins. In this technique a mixture of proteins is
separated based on molecular weight, and through gel electrophoresis. Then the results are
transferred to a membrane producing a band for each protein. The membrane is then incubated
with labels antibodies specific to the protein of interest (Mahmood and Yang 2012). Western
blotting is also used in the detection of circulating antibodies specific to a single protein or
several proteins, in gene expression studies and in the analysis of biomarkers such as hormones,
growth factors and cytokines (Cheriyedath 2018). Proteins adhere to the membrane in the same
pattern as they have been separated because of interaction of charges. These proteins on this
immunoblot are then ready for antibody binding for detection. Antibodies are used to detect
proteins on the western blot also known as immunoblot. This process is known as blotting
(Antibodies online-.com 2013).

Conclusion
In a nutshell, it can be concluded that from the hydropathy plot of protein sequence 1 the
receptor protein has characteristics of a G-protein coupled receptor (GPCR). For protein
sequence 2 , the receptor protein has the characteristic of gamma-aminobutyric acid type B
receptor subunit 2 (GABBR2). The molecular weight of protein sequence 1 is 47402.08 and for
protein sequence 2 is 111483.66. The pI value of protein sequence 1 and 2 are 6.7114 and 8.7198
simultaneously. Furthermore, by using the computational analysis, we have determined the
unknown nucleotide sequences as the growth hormone-releasing hormone receptor (GHRH-R).
It is a releasing hormone of growth factor (GH) which is highly expressed in human
retinoblastoma (RB) cells, but not in other retinal cells. It is a 44 amino acid peptide bond
produced in the arcuate nucleus of hypothalamus. Moreover, GABBR2 had identified from the
unknown sequence by the computation analysis. It belongs to GABA-B receptor subfamily,
enable inhibit neuronal activity thereby control the regulation of the neurotransmitters. It is
activated by forming heterodimeric complex with GABA-B receptor subunit 1.

The similarity between this two protein sequences is only 20.5%, therefore it is simple to
differentiate. The most likely tests to qualitatively detect and confirm the presence of the two
receptors are immunofluorescence and SDS-PAGE. These methods were applied due to their
high specificity for the differentiation of similar proteins is almost absolute, barring any cross-
reactivity between the species. The applications of this practical and also the research area play
an important role for the future of science.

References
● Mahmood, T & Yang, CP 2012, ‘Western Blot : Technique, Theory and Trouble
Shooting,’ North American journal of medical sciences, vol 9, no 4, pp 429-434.
● Caprette, DR 2012, ‘Experimental Biosciences, Resources for introductory &
intermediate level laboratory courses: Introduction to SDS-PAGE’, viewed 21 June 2018,
<http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html>.
● Cheriyedath, S 2018, western blot -lab techniques used to detect proteins, viewed 21 june
2018, <https://www.news-medical.net/life-sciences/Western-Blot-Lab-Technique-Used-
to-Detect-Proteins.aspx>.
● News Medical 2014, What is Flow Cytometry,viewed 22 June 2018, < https://www.news-
medical.net/life-sciences/What-is-Flow-Cytometry.aspx> .
● Flow Metric 2018, What is Flow Cytometry, viewed 22 June 2018, <
https://www.flowcytometryservices.com/cytometry-blog/what-is-flow-cytometry> .
● Svar Life Science 2018, Immunofluorescence, viewed 22 June
2018,<http://www.eurodiagnostica.com/index.php?headId=3&pageId=3&langId=1&catI
d=10>.
● Antibodies online.com 2013, western blotting ; gel electrophoresis for proteins, viewed
22 june 2018, < https://www.antibodies-online.com/resources/17/1224/western-blotting-
immunoblot-gel-electrophoresis-for-proteins/>.