University of Minnesota Duluth 2014 UROP paperSwenson College of Science and Engineering, University of Minnesota Dulut

Schramel, Stephanie

University of Minnesota Digital Conservancy

Duluth Journal of Undergraduate Research 2014 
 
108 
Structure – Function Studies of Annexin A2 & A5 
 
Stephanie Schramel 
Swenson College of Science and Engineering 
University of Minnesota Duluth 
Schra348@d.umn.edu 
 
 
1. Introduction 
 
Within the cell, proteins exist to maintain the intracellular level of calcium ions (Ca
2+
), allowing 
calcium to act as a secondary messenger. Annexins are a group of these proteins located in 
muscle cells which bind calcium in order to bind to phospholipids in the plasma membrane. 
Although there are multiple forms of this protein found in humans, all contain a similar structural 
basis consisting of an amino-terminal head domain, a carboxyl terminal domain, and conserved 
structural repeats of around 70 residues. Both the calcium binding and membrane binding sites 
are located at the carboxyl domain. Annexins bind to negatively charged phospholipids at the 
cellular membranes, which differentiates them from other calcium binding proteins. 
Exocytosis/endocytosis, ion transport, and transport of vesicles are also processes orchestrated 
by these proteins.
1
   
 
2. Research Methodology and Findings 
 
Specifically, annexin A2 (AnxA2) and A5 (AnxA5) are the proteins we studied. Figure 1 shows 
the 3-D structure of AnxA2 with calcium bound. The question we tried to understand is the 
mechanism for how AnxA2 and AnxA5 function at the membrane in terms of the redistribution 
of lipids and the effect on membrane permeability. In order to study the annexins, we grew E. 
coli cells that expressed our desired proteins. We then went through multiple week-long 
purification processes to obtain pure protein to be used for further studies. In the case of AnxA5, 
we were able to successfully purify the protein using a known protocol. For AnxA2, a 
purification protocol was not known; therefore one was created based on the knowledge from the 
AnxA5 protocol. Two purifications were completed, but neither was successful. Since AnxA2 is 
a membrane binding protein, it was hypothesized that the protein bound to dialysis tubing during 
the dialyzing step to filter out unwanted proteins and other molecules. Currently, we are trying to 
modify the protocol so that a successful purification can take place. 
  
Duluth Journal of Undergraduate Research 2014 
 
109 
Figure 1. Structure of Annexin A2 bound 
to calcium.2 Calcium is shown as the 
black spheres attached to the α-helicies. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In order to get an understanding of the mechanism of the annexin A5, structural and stability 
experiments were done. These experiments include differential scanning calorimetry (DSC), 
isothermal titration calorimetry (ITC), and fluorescence lifetime spectroscopy (FLT). DSC was 
used to determine the thermal stability and heat capacity of proteins. ITC was used to determine 
the change in enthalpy and binding affinity for the interaction of molecules. FLT used 
fluorescence as a way to study biochemical interactions of the protein.  
 
Figure 2 shows data obtained using FLT for annexin A5 to give information on the effects of 
membrane permeability. As shown, the efflux of carboxyfluorescein (CF), a dye which was 
monitored by the fluorescent signal given off, decreased with AnxA5 present. Therefore, annexin 
A5 has a direct impact on reducing the membrane permeability. The presence of cholesterol also 
decreased the efflux of (CF), meaning the presence of cholesterol and annexin prevents 
movement across the membrane.
3
 
Duluth Journal of Undergraduate Research 2014 
 
110 
 
 
 
 
 
 
 
 
 
 
 
3. Conclusion 
 
Other studies have looked at the binding mechanism of Ca
2+
 with AnxA5 at the membrane to test 
for cooperative binding. Cooperativity would occur if the binding of one calcium ion increased 
the binding of more calcium ions. It was shown that AnxA5 can be found in two states: the first, 
annexin can be free in solution, and the other can be membrane associated. The membrane 
associated state has higher affinity for calcium than does the solution state. Cooperative binding 
of calcium only occurred if there was a low concentration of AnxA5 in the membrane bound 
state with no calcium initially present. Otherwise, the binding of calcium was found to be 
independent.
4 
 
From here, our goal is to modify the annexin A2 purification protocol so that further studies can 
be run on this protein, as well as A5.  
 
 
  
Figure 2. The efflux of carboxyfluorescein (CF) from Large Unilamellar Vesicles composed of 
POPC:POPS (60:40) and varying cholesterol. All samples contained 333 μM lipid and all plots are 
an average of three trials. Black: lipid alone; blue: lipid and Ca2+; pink: lipid and Annexin a5; 
purple: lipid, Annexin a5, and Ca2+. (A) POPC:POPS (60:40), no cholesterol in the vesicles; (B) 
POPC:POPS (60:40), with 5% cholesterol; (C) POPC:POPS (60:40), with 10% cholesterol; (D) 
POPC:POPS (60:40), with 20% cholesterol; (E) POPC:POPS (60:40), with 30% cholesterol; (F) 
POPC:POPS (60:40), with 40% cholesterol. The inset in (F) shows the decrease in temperature for 
all scans over 60 minutes with the water bath set to decrease from 20°C to -3°C.3 
Duluth Journal of Undergraduate Research 2014 
 
111 
References 
 
Bharadwaj, A.; Bydoun, M.; Holloway, R.; & Waisman, D. (2013). Annexin A2 Heterotetramer: 
structure and function. International Journal of Molecular Sciences, 14(3), 6259-305. 
 
Gauer, J. W.; Knutson, K. J.; Jaworski, S. R.; Rice, A. M.; Rannikko, A. M.; Lentz, B. R.; & 
Hinderliter, A. (2013). Membrane modulates affinity for calcium ion to create an apparent 
cooperative binding response by Annexin A5. Biophysical Journal, 104(11), 2437-47. 
 
Ranniko, Anika. (2013). The Annexin-Lipid party line: Communication between protein and 
membrane. A presentation at the 27
th
 Annual Gibbs Conference on Biothermodynamics (October 
5-8, 2013 at the Touch of Nature environmental Center, Southern Illinois University, 
Carbondale, IL.) 
 
Shao, Chenghua; Zhang, Fuming; Kemp, Melissa M.; Linhardt, Robert J.; Waisman, David M.; 
Head, James F.; & Seaton, Barbara A. (2006). Crystallographic analysis of calcium-dependent 
heparin binding to Annexin A2. The Journal of Biological Chemistry, 281(42), 31689-95. 
doi:10.1074/jbc.M604502200 
 
 
 
Project Faculty Adviser: 
Dr. Anne Hinderliter, Department of Chemistry and Biochemistry, University of Minnesota 
Duluth. Email: ahinderl@d.umn.edu 
 
 
 
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Structure – Function Studies of Annexin A2 & A5

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