Phosphoserine-rich Protein from Phragmatopoma californica. Leila Jirari Mentor: Chengjun Sun Herbert Waite NIH, NSF, NASA

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Alberta Gibbs
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1 Phosphoserine-rich Protein from Phragmatopoma californica Leila Jirari Mentor: Chengjun Sun Herbert Waite NIH, NSF, NASA

2 Why Study This Worm? Underwater adhesive Glue adheres to eggs shells, glass, teeth Find out HOW adhesion happens under water

3 Distribution: Europe 3, East coast 2, California 1, Indian Ocean 1 Tube Worm Phylum Annelida Class Polychaeta Order Terebellida Family Sabellariidae Derivation: Sabell-sand(L); -aria - to dwell "sand dweller"

5 Hold On

6 Phragmatopoma californica colony

7 2 mm

8 Cement gland Pc-1 Pc-3 Pc-2

9 Tube growth with silica beads

10 Foam Skin

11 Proteins Known in Glue Glue has over 25 mol% of serine. Pc-1, Pc-2 both glycine rich. Proteins were purified in the 80 s. Post-translational modification: tyrosine to dopa. Pc-3 Serine rich. Deduced from cdna sequence, protein has not been purified. Post translational modification: serine to phosphoserine.

12 NH-CH-CO CH 2 NH-CH-CO CH 2 OH OH HO OH P = O OH Tyrosine Serine

13 NH-CH-CO CH 2 NH-CH-CO CH 2 OH OH HO O P = O OH Dopa Phospho-serine *Dopa: 3,4 dihydroxy-phenylalanine

14 Worm Glue Protein Extraction Aim: To purify pc-3 Serine-rich protein (60-90% serine) or peptide (glue) Problem: Not extractable from glue Try from glue: (1) Protein extraction (from previous work, didn't do) (2) Partial Acid Hydrolysis to break up protein Try from thorax where all glue proteins were made: (1) Protein extraction w/ 5% Acetic acid followed by CaCl 2 precipitation (2) Protein extraction w/ 0.2M Tris w/ EDTA followed by CaCl 2 precipitation

15 Methods: Flow Chart 1 Partial acid hydrolysis Worm glue &6M HCl Heat for 5 min@100c Run P-ser column: gets peptides w/ P-ser b/c will bind to FeCl 3 in column Elution w/ ammonium bicarbonate buffer and aaa no P- ser rich protein Flow through aaa no P- ser rich protein

16 mau ADC1 A, ADC1 CHANNEL A (030401\TEST4958.D) min glu pro cys val met ile leu dopa phe his hylys lys arg asp ser thr Results: Flow through gly ala mau % serine P-ser column flow through ADC1 A, ADC1 CHANNEL A (030401\TEST4977.D) min pro cys val met ile dopa leu tyr phe hylys lys asp thr ser glu gly his Results: Bound to P-ser column bound 7% serine

17 Methods: Flow Chart 2 Worm thorax (5% Acetic acid supernatant) (1)Run P-ser FeCl 3 column 1:4 CaCl 2 Precipitant (2) Take supernatant final [CaCl2] 1:2 aaa on precipitate no P-ser rich protein aaa on precipitate some P-ser rich protein Elution w/ ammonium bicarbonate freeze dry and aaa no P-ser rich protein Flow through aaa no P-ser rich protein

18 AU ADC1 A, ADC1 CHANNEL A (030401\TEST5032.D) glu (1) P-ser column elution asp ser gly 7% serine thr ala val met ile leu phe his lys arg min mau ADC1 A, ADC1 CHANNEL A (030401\TEST5012.D) asp ser glu (2) CaCl 2 Precipitate 8.5% serine & P-ser 1000 ala 800 thr 600 leu lys val met ile dopa phe his hylys

19 Methods: Flow Chart 3 Worm thorax 0.2M Tris buffer w/ 0.2M EDTA, ph 7.4 Homogenize & centrifuge Collect supernatant Collect pellet Homogenize in 5% Hac & 8M Urea 3 1 Add CaCl 2 to final concentration of 1M Dialyze (supernatants) extract against H 2 O aaa 2 1. Some serine protein present (10 mol % serine) 2. No high serine protein 3. No high serine protein

20 1. Tris/EDTA extract dialyze against H 2 O precipitant mau ADC1 A, ADC1 CHANNEL A (030401\TEST5060.D) asp thr ser glu gly ala Results: 11% serine val ile leu dopa tyr phe his hylys lys min

21 2. AU extract after Tris-EDTA, dialyze against H 2 O precipitant mau ADC1 A, ADC1 CHANNEL A (030401\TEST5061.D) asp glu Results: 7.5% serine thr ser ala 1000 val ile leu tyr phe his lys min

22 3. CaCl 2 Precipitate from dialyzed Tris/EDTA extract mau ADC1 A, ADC1 CHANNEL A (030401\TEST5046.D) asp Results: 8.6% serine thr ser ala 600 leu val ile tyr phe his lys mi

23 Next Keep looking! Redo Tris/EDTA extraction and dialyze against water. Analyze the precipitate. Redo the 5% HAc protein extraction followed by CaCl 2 precipitation and use a higher concentration CaCl 2.

24 What I have learned Save & label everything! Little marine tube worms have perfected synthesis and secretion of an under water adhesive that has taken years to learn about and there is still so much unknown Research can be so exciting and also so repetitive.

25 Thank You!

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