Transcript Document

Chemical/thermal denaturation of Alkaline
Phosphatase
Allen Suarez
Juliana Liang
William Pinkston
In collaboration with:
Allison Morley
Blake Tye
Duyen Vo
Biochemistry 463a
Spring 2012
Abridged conclusion:
Alkaline phosphatase = always in beast mode
Come at me,
bro!
PDB ID 1ED8
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 phosphatase1.
• However, last Spring 2011, a student group
showed that AP denatures near 90°C using a
circular dichroism method2.
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 temperature4, 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.
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 mechanism6.
• 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 intraprotein interactions (indirect effect)7.
from http://andromeda.rutgers.edu/~huskey/335f09_lec.html
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)
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.
What are we looking for in a CD spectra?
Ellipticity,
can also be
expressed
as θ (molar ellipticity)
In units of
degcm2/dmol
Alkaline Phosphatase
Secondary Structure
17 α-helices:12 β-strands: 6 β-turns per monomer
Dimer shown
PDB 1ED8
Circular Dichroism – the instrument
from http://www.olisweb.com/literature/pdf/cd_practical_guide.pdf3
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
Circular Dichroism – Scan
0
-2000200
210
220
230
240
250
-4000
[θ] (deg x cm2 x dmol-1)
-6000
-8000
-10000
-12000
-14000
-16000
-18000
-20000
AP in Buffer - 25C
-22000
AP in Urea - 80C
-24000
-26000
-28000
-30000
λ (nm)
260
Circular Dichroism – Melt Curves
0
70
75
80
85
90
95
100
-2000
% Helicity
[θ] (deg*cm^2/dmol)
-4000
Sample
PreDenatu
ration
PostDenatu
ration
AP in
Buffer
16.4%
@ 90°C
5.2% @
96°C
AP in
8M
urea
37.2%
@ 80°C
14.8%
@ 94°C
-6000
-8000
-10000
AP in Urea
AP in Buffer
-12000
-14000
-16000
Temperature (°C)
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 Tm 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.
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?
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.
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).