Transcript Slide 1

Bee Venom Lab
Anu Murphy
Introduction
• We will use web-based biological tools to study
various bee venom toxins.
• Outline of presentation:
– Which toxins are present in bee venom? Obtain
information from the NCBI databases
– Obtain and manipulate amino acid sequences of
toxins using the Biology Workbench (BW)
• Note to teachers:
• (a) In order for this tutorial to work properly, please make sure that the popup blocker on your computer is disabled.
• (b) For protein visualization, it is necessary to download Cn3D, a structure
viewing program (available for free) at:
http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dinstall.shtml
Terms you need to know:
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Erythrocyte: A red blood cell (RBC). Hemoglobin molecules contained
within the RBC help carry oxygen around the body.
Hemolysis: Breakdown of an RBC cell wall resulting in the release of
hemoglobin molecules.
Lipid bilayer: A double layer of lipid (fat) molecules. Proteins, cholesterol,
and phospholipid molecules (amphipathic molecules containing a charged
region at one end and fatty acid chains at the other) are the major
components of cell walls.
Trp19: The amino acid tryptophan at position 19 of the melittin sequence.
Amino acid residues: Building blocks of proteins. 20 amino acids exist in
nature, from with all proteins are made. The order and frequency of
recurrence of different amino acids within a protein sequence is determined
by its DNA sequence.
Conservation among amino acids: The degree of similarity between two or
more protein sequences can be an indication of similarity in their structure
and/or function. In order to determine such similarities, we examine how
closely the protein sequences resemble each other. If the amino acids
present at each position along the protein sequences are either identical or
very similar (i.e., in terms of size and polarity of the amino acids) then we
say they are highly conserved. If the amino acids are very different, they
are said to be weakly conserved.
Using the NCBI databases
• The NCBI website at http://www.ncbi.nlm.nih.gov/
is an online resource for conducting biological research.
Here you will find many easy to use databases, including
Pub Med (database of biological literature), GenBank
(database of genetic sequences), Structure (database of
macromolecular 3D structures), and BLAST (search tool
for genetic sequences)
• Click on Pub Med to access the literature database; start
a search for the keywords “bee venom toxin”
• Looking through the results of the search, we find that
melittin, apamin, tertiapin, and MCDP are well known
toxins present in bee venom that affect human cells
NCBI databases contd ..
• A Pub Med search on melittin yields information on many of its
properties: (explanation of the underlined terms can be found in the
Glossary section on page 14 of this presentation)
– Melittin causes cell lysis (I.e., ruptures the cell membrane) in
erythrocytes; presence of cholesterol in the erythrocyte membrane
inhibits hemolysis (Raghuraman H & Chattopadhyay A. Chem. Phys
Lipids. 2005 Apr;134(2):183-9)
– Results of mutation studies on folding and insertion of melittin in lipid
bilayers
– Trp-19 is significantly involved in the cell membrane rupturing activity of
melittin (Blondelle SE, Simpkins LR, Perez-Paya E, Houghten
RA.
Biochim Biophys Acta. 1993 Oct 6;1202(2):331-6)
– Effect of melittin on ion transport across cell membranes: melittin
interacts with negatively charged phospholipids and inhibits transport
pumps in the cell membrane (Yang S, Carrasquer G.
Zhongguo Yao Li Xue Bao. 1997 Jan;18(1):3-5.
– Effects of melittin on arthritis patients (Park HJ et al. Arthritis
Rheum. 2004 Nov;50(11):3504-15)
NCBI databases contd …
• For a preliminary look at the structure of melittin, go back
to http://www.ncbi.nlm.nih.gov/ and click on “Structure” at
the top menu bar.
• In the Structure page enter 1BH1 in the search window
and click on “Go” (1BH1 is the accession number for
melittin in the PDB database)
• In the results page for Structure, click on the link to 1BH1
• In the MMDB Structure Summary page click on “View 3D
Structure” of “Best Model” with “Cn3D” (you have to
select this option in the window), “Display”. In the pink
graph just below this note protein chain length is 27
amino acids.
• The 3D structure of Melittin appears on the screen. Click
on the right mouse button for further display options
Using the Biology Workbench
• BW is available at:
– http://workbench.sdsc.edu/
– click on “Set up a free account” to register
– click on “Enter the Biology Workbench 3.2”
Using BW Contd. ...
• In the menu at the bottom of the page, click on
“Session Tools”
• To name your session:
– In the Session Tools menu click on “Rename Session”
and “Run”; then type in “Bee venom Analysis”, and
click on “Submit”
• To perform a search for protein sequences:
– Click on “Protein Tools” at the top of the page
– Select “Ndjinn – Multiple Database Search” from the
menu and click on “Run”
Using BW contd …
– In the Ndjinn page search box, type “melittin”, select
50 “Hits per page” and Full “Display mode”, scroll
down, then click on boxes for the following protein
sequence databases: PDBSEQRES, SDSCNR, and
SWISSPROT
• Note: The above three databases were selected for the following reasons:
– SDSCNR is the non-redundant database of all protein sequences
contained within the Biology Workbench
– SWISSPROT is an independent, exhaustive biological database
containing many unique and useful tools in an easy-to-use format
– PDBSEQRES contains all biological sequences with reported 3-D
structural data
– Click on “Search” near the top of the page
– Your search results will show up shortly (this may take
a few minutes)
Using BW Contd. …
• To view and manipulate protein sequences:
– From the search results, select the following melittin sequences :
SDSCNR:481966 (Melittin, major – Indian honeybee, 26 AA),
SDSCNR:1605225 (P01504 Melittin, 26 AA), SDSCNR:495401 (Melittin
minor – honeybee, 27 AA), SDSCNR:3200772 (P01502 Melittin, 26 AA),
PDBSEQRES:1BH1 (toxin). Avoid sequences described as “Melittin
precursor”, “Melittin resistance protein”, “Melittin signal peptide”, etc.
– Select a few other sequences that interest you. Make sure the
sequence descriptions include the term “melittin” and sequence length
is around 26 amino acids.
– Click on the “Import Sequences” button at the bottom menu of the
search results page. Selected protein sequences will appear in the Bee
Venom Analysis session page.
– In the Bee Venom Analysis page, select protein sequences by clicking
on the respective boxes, click on “View Database Records of Imported
Sequences”, in the menu box, and “Run”
– Select Database Format: “Formatted” in the View Records page, then
click on “Show Records” (this may take a few minutes)
Using BW Contd..
– Upon examination of the protein database records, we find that reported
sequences of melittin contain 26-27 amino acids
– Melittin has one record in the PDBSEQRES database, which also
contains 3D structural information for the protein
• To view the structure of melittin:
– In the “View Records” page scroll down to the PDBSEQRES database
record for melittin (under PDBSEQRES: 1BH1) and click on “View 1bh1
Structure” on the right hand side of the record. A 3-D structure of
melittin will show up shortly. Within the structure viewer window, click
on the left mouse button to manipulate the protein, right mouse button
for more display options
– Note: in the PDBSEQRES record page for 1BH1 you can also click on
“PDB Structure Explorer” to get a more interactive 3D display of melittin.
Within the PDB Structure Explorer you will find freeware molecular
structure viewer programs available for downloading
Using BW Contd…
• To perform protein sequence alignments:
– At the bottom of the View Records page, click “Return” to return to the
Protein Tools page in BW. Select all melittin sequences
– Click on “CLUSTALW - Multiple Sequence Alignment” in the menu, then
“Run”
– Accept default parameters and click “Submit” in the CLUSTALW page
– Examine sequence alignments produced in the CLUSTALW results
page; note strong conservation of amino acid residues within the
protein. TRP-19 (designated by the symbol “W” at position 19 in the
melittin sequence) is conserved throughout all the sequences (this is
important because TRP-19 plays a crucial role in the proper functioning
of melittin)
– Scroll down further to check out the Unrooted tree generated by the
alignment program. This gives you some idea of the evolutionary
distances between the melittin sequences that were selected
– Print results
– Click on “Import Alignment(s)” near the bottom of the page. This will
take you to the Alignment Tools page
BW Contd…
– Select the CLUSTALW alignment by checking on the
corresponding box, select “BOXSHADE – Color Coded Plots of
Pre-Aligned Sequences” from the menu and click on “Run”
– Accept default parameters and click on “Submit” in the
BOXSHADE page
– Examine the color-coded alignment plot. Amino acid residue
variances between the sequences are clearly indicated by color:
green for identity among amino acids at a certain position, yellow
and blue for partial identity, and gray for complete dissimilarity
– Print results
– Click on “Return”
BW Contd …
• To draw a phylogenetic tree:
– In the Alignment Tools page menu select “DRAWTREE – Draw
Rooted Phylogenetic Tree from Alignment”, click on the box by
the CLUSTALW alignment to select the alignment, click on “Run”
– Accept default values in the DRAWTREE page and click on
“Submit”
– The resulting tree produced from our alignment of melittin
sequences illustrates the evolutionary distances between your
protein sequences
– Print results
• Note: since the melittin sequences selected for this exercise were
very similar, the evolutionary tree produced from their alignment is a
relatively simple one.
– Return to the Alignment Tools page. Try out different menu
options to manipulate your sequence alignment
• Repeat the above exercise with apamin and tertiapin, both well
known toxins present in bee venom. Start with a preliminary
literature search on the Pub Med database at
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed to obtain
relevant functional and structural data. Follow up with a more
detailed study on the Biology Workbench at
http://workbench.sdsc.edu , where you can:
(a) Obtain and view protein sequences of apamin and tertiapin
(b) view 3-D structures of proteins (if available)
(c) construct sequence alignments to examine similarities and
differences at regions of interest throughout the proteins
(d) construct phylogenetic trees to study evolutionary history of the
proteins
Questions for discussion
• After completing the tutorial in this presentation for
melittin, go through the same steps for apamin. Then,
– Compare and contrast how these two bee venom toxins affect
human cells.
– Examine phylogenetic trees of melittin and apamin. How do they
compare?
• Look through the web sites on bee venom therapy
(included in the Bee Venom Inquiry Unit, in the list of
web sites). Do you think bee venom therapy is a valid
method of treatment for arthritis and multiple sclerosis
(MS)? Why or why not?
Acknowledgements
• Funding for this project was provided by
the Water CAMPwS, a National Science
Foundation Science and Technology
Center.