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 pop-

up 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:

• 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.