1. Biochemistry 463a Spring 2012 Chemical/thermal denaturation of Alkaline Phosphatase Allen Suarez Juliana Liang William Pinkston In collaboration with: Allison Morley Blake Tye Duyen Vo

2. Abridged conclusion: Alkaline phosphatase = always in beast mode Come at me, bro! PDB ID 1ED8

3. Past Experiments In past studies of the denaturation of AP students have used 8M urea at room temperature and saw no significant unfolding of the phosphatase 1. However, last Spring 2011, a student group showed that AP denatures near 90°C using a circular dichroism method 2.

4. Goals & Hypothesis We set out to denature AP using 8M urea at an elevated temperature in order to assess the stability of this enzyme using the circular dichroism method of analyzing secondary structure. Initial hypothesis: Given that 8M urea denatures most proteins even at room temperature 4, 5, we predict denaturation of AP will occur somewhere between ~40°C - 55°C in the presence of 8M urea. Revised hypothesis: Because no significant denaturation was observed in our initial range, we predict AP will denature after 30 minutes in 8M urea at ~70°C. Further revised hypothesis: After observation that no significant unfolding occurred at 70°C, we now predict denaturation will occur in 8M urea at 80°C.

5. Urea - the denaturant Uncharged – implicates its interaction with hydrophobic groups of proteins Originally thought to act in a similar manner as GdnHCl, but was found to denature proteins via a separate mechanism 6. Forms non-covalent interactions with peptide backbone, stabilizing non-native (i.e. unfolding intermediates) structures (direct effect) 7. Disrupts water-water interactions, making water more able to solvate hydrophobic groups, allowing water to compete with intra- protein interactions (indirect effect) 7. from http://andromeda.rutgers.edu/~huskey/335f09_lec.html

6. Circular Dichroism Principles UV circular dichroism measures the differential absorption of either left or right circularly polarized light by a chiral (asymmetric), UV absorbing molecule (i.e. the peptide backbone of alkaline phosphatase)

7. Circular Dichroism Principles Ordered structures such as alpha-helices and beta sheets display characteristic spectra between 260 nm and 200 nm. This principle is key to monitoring secondary structure of proteins and thus unfolding.

8. What are we looking for in a CD spectra? Ellipticity, can also be expressed as θ (molar ellipticity) In units of deg  cm 2 /dmol

9. Alkaline Phosphatase Secondary Structure 17 α-helices:12 β-strands: 6 β-turns per monomer Dimer shown PDB 1ED8

10. Circular Dichroism – the instrument from http://www.olisweb.com/literature/pdf/cd_practical_guide.pdf 3

11. Methods We measured: - The extent of denaturation of alkaline phosphatase (85.8 μM stock) from Sigma Aldrich in 8M urea at various temperatures using the Olis DSM 20 CD instrument in Bioscience West. Temperatures: Scan of AP in Tris HCl pH 7.36 @ RT (control) Scan of AP in Tris HCl pH 7.36 w/ 8M urea after 30 min. @ 80°C Melt curve of AP in Tris HCl pH 7.36 from 50°C-98°C (control) Melt curve of AP in Tris HCl pH 7.36 w/ 8M urea from 70°C-98°C [AP] = 0.312 mg/mL for all conditions

12. Circular Dichroism – Scan

13. Circular Dichroism – Melt Curves SamplePre- Denatu ration Post- Denatu ration AP in Buffer 16.4% @ 90°C 5.2% @ 96°C AP in 8M urea 37.2% @ 80°C 14.8% @ 94°C % Helicity

14. Conclusions 8M urea appears to contribute insignificantly to the denaturation process of Alkaline Phosphatase, even at temperatures above 80°C. The only effect 8M urea has on the melt curve of Alkaline Phosphatase compared to a solution containing no urea is to lower the T m value by less than 5°C, indicating heating to these temps is sufficient to denature without chemical denaturants. Interestingly, we found the AP in urea produced larger negative values for θ than AP in only buffer. This indicates the enzyme was “more folded” in urea than when no urea was present.

15. Further Questions If time is a factor in the chemical denaturation of proteins, will AP denature completely in urea when it is allowed to incubate for longer than 30 minutes (i.e. 2 hours? 3 hours? Etc.)? What is the reason that AP in urea is “more folded” than AP only in buffer?

16. Acknowledgements We would like to thank our collaborative group of Blake Tye, Duyen Vo and Allison Morley for their patience and aide in searching for an appropriate temperature to run these experiments. We would especially like to thank Dr. Chad Park for his excellent assistance and guidance in using the CD instrument. Last, but not least, we would like to thank Kayla, Nicole, Swapna and Dr. Hazzard for their advice (and constructive criticisms) throughout the arduous process of attempting to denature the beast.

17. References 1. Boyadjian, N., Childers, K., Luiten, R., and O’Neil, L. “Circular Dichroism of Alkaline Phosphatase” (lab report for BIOC 463a), 2011. 2. Louis, A., Mehlau, M., Nelson, M., and Sund, D. “Circular Dichroism” (lab report for BIOC 463a), 2010. 3. Copeland, R.A. Methods for Protein Analysis: A Practical Guide; Olis, Inc.: Georgia, 1994; p 3. 4. Pace, C. N. Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves. Methods Enzymol. [Online] 1986, 131, 266-280. http://www.biochem.arizona.edu/classes/bioc463a/special_res_proj/urea_gdnhcl_pace86.pdf (accessed Apr 20, 2012). 5. Pace, C.N., and Shaw, K.L. Linear Extrapolation Method of Analyzing Solvent Denaturation curves. Proteins: Struct., Funct., Genet. [Online] 2000, 4, 1-7. http://www.biochem.arizona.edu/classes/bioc463a/special_res_proj/solvent_denaturation_pace_shaw.pdf. (accessed Apr 20, 2012). 6. Monera, O.D., Kay, C.M., and Hodges, R.S. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. [Online] 1994, 3 (11), 1984-1991. http://onlinelibrary.wiley.com/doi/10.1002/pro.5560031110/abstract;jsessionid=8218EC6F8BA4F6409948549627E1D4 BE.d03t03. (accessed Apr 23, 2012). 7. Bennion, B.J., and Daggett, V. The molecular basis for the chemical denaturation of proteins by urea. Proc. Natl. Acad. Sci. U.S.A. [Online] 2003, 100 (9), 5142-5147. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154312/ (accessed Apr 23, 2012).