Calcium Binding Properties of Fibulin-5 Jan Sterniczuk Dr. Smith Lab
The Fibulin Family Timpl R., Sasaki T., Kostka G., Chu M. (2003)  Nature  4,   479-489
<ul><li>Notch-1  </li></ul><ul><li>Comprises 40 residues with three invariant disulfide bonds paired 1-3, 2-4 and 5-6 orie...
<ul><li>Vitamin-K dependent protein S </li></ul><ul><li>Not always elongated </li></ul><ul><li>variation in structure, mod...
Structure and Function of Fibulin-5   RGD <ul><li>Predominantly expressed in developing arteries   (Nakamura 1999) </li></...
Fibulin-5’s cbEGFs proposed Ca 2+  binding Epidermal Growth Factor Modules Extended C-terminal Region RGD Does fibulin-5 b...
Thesis Objectives <ul><li>To elucidate the nature of calcium binding to fibulin-5. </li></ul><ul><ul><li>Purify and refold...
Protein Derivatives of Fibulin-5 448 a.a. 351 5 6 264 344 EGF-A 5 6 264 EGF-B SP 2 3 4 5 6 1 N C
Experimental Procedure Protein Constructs  Protein Expression (Bacterial System) Refolding in redox buffer  – cysteine/cys...
Expression and purification of  and Expression of EGF-B at 37 °C std. uninduced induced A A Ni 2+  elution B Ni 2+  elutio...
Purification of    (Size exclusion chromatography) 1 2 3 5 6 EGF-B 20 25 std 1 2 3 15
CD analysis of calcium-binding to 5 6 EGF-B μ M μ M Ca 2+
CD analysis of   5 6 EGF-A
CD Spectra of other EGF-like  modules Recombinant cbEGF 7-9  microneme protein 4  Periz et al, Molecular & Biochemical Par...
Experimental Procedure Protein Constructs  Protein Expression (Bacterial System) Refolding in redox buffer  – cysteine/cys...
15 N Labeling <ul><li>Growth of cells on minimal media </li></ul><ul><ul><li>Grow cells in salts and minimal nutrients </l...
2D  1 H- 15 N-HSQC Backbone amide nitrogen  Simplistic Spectrum Polypeptide 15 N 1 H H 15 N R.f. Pulse Relaxation
2D  1 H- 15 N HSQC NMR analysis of the folding and Ca 2+ -binding properties of   No Ca 2+ 5 6 EGF-B 10mM Ca 2+ 5mM Ca 2+
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Calcium Binding Properties Of Fibulin 5

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Undergraduate thesis presentation on the three dimensional folding properties of a multi-domain Fibulin protein.

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  • The fibulins family is made up of six extracellular matrix glycopoteins and are found in a variety of tissues but are predominantly found in the vasculature. They function as scaffold and adhesion proteins. They associate with a diverse array of supramolecular structures, including elastic fibers, basement membrane networks, fibronectin microfibrils and proteoglycan aggregates. Mutations in the fibulin gene are associated with heritable diseases. For example mutations in fibulin-5 result in cutis-laxa which is a disease that is characterised by loose and sagging skin. This is because fibulin-5 is intimately associated with proper elastin formation. As we can see from the image above the N-terminal varies immensly and this variation is responsible for the variety of function of the fibulins. The C-terminal domain of is conserved throughout the fibulins. The C-terminal domain doesn’t show any secondary structure. It is this C-terminal domain that differentiates the various fibulins since there C-terminal domains have a variety of ligands. For example Fibulin-5 can bind superoxide dismutase and Lp(a). There are also splice isoforms that result in varying lengths of the C-terminal domain as is the case of fibulin-1. Of particular interest is the Epidermal-Growth Factor domain that is conserved and found in multiple repeats. A subset of the EGF-like domains bind calcium which provides the fibulin with structural stability. We are specifically interested in fibulin-5.
  • The calcium binding EGF-like module is found in a variety of extracellular matrix proteins for example protein S, factor IX, and the fibrillin family, notch family the LDL receptor and of course the fibulin familiy . The cbEGF domain is comprised of 40 residues with three invariant disulfide bonds paired 1-3, 2-4 and 5-6 orientation. The calcium binding consensus sequence is located at the N-terminal. cbEGF domains don’t exhibit many different types of secondary structural elements, but do have several antiparallel β-sheets. Upon binding to calcium the EGF is stabilized into a rod-like conformation. Notch-1, comprising EGF-like domains 11–13
  • Fibulin-5 is a 448 amino acid protein that was first characterized by Nakamura and colleagues in 1999. This intergrin binding extracellular matrix protein is found in atherosclerotic lesions and is intimately associated with elastin formation. integrins [alpha v beta 3] and [alpha v beta 5] It is made up of six EGF domains and an extended C-terminal domain. These EGF can bind calcium. RGD – Argenine-Glycine –Aspartic acid cell-adhesion motif Lp(a) interaction. C-terminal domain binds apo(a)’s KIV2 sequence.
  • Structural studies of these cbEGF domains have been routinely performed on modular pairs. It has been shown that the tandemly repeated cbEGF domains are structurally stabilized in the presence of calcium. This is why we have adapted a similar strategy. to use as a tag to solubilize the C-terminal region. We have been working on elucidating the nature of calcium binding to the 5 th and 6 th EGF modules of fibulin-5. Of primary importance to this study is the expression and purification of the 5 th and 6 th EGF modules with varying lengths of the C-terminal region of Fibulin-5. Truncation were made prior to my arrival.
  • There are ten truncations and we are currently working with constructs A and B. We have adopted a dissecting approach to our research. By properly purifying and refolding the smallest constructs A that contains twelve cysteins and B that contains fourteen cysteins it is our belief that the purification and refolding technique can be used for our other constructs. This methodology will allow us to determine if the calcium binding properties are a function of the 5 th and 6 th EGF modules and not of the C-terminal domain.
  • redox buffere is to
  • Beta sheet character usually minimum around 215nm.
  • pI B construct  4.88 This data suggests that that we have properly folded protein. We The purification and refolding conditions were repeated but the proteins were grown up in N15 labelled media. Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells.
  • redox buffere is to
  • Correlation spectrum. N15 on Y-axis and H1 on the X-axis. numbre of peaks not same as 87 amino acids. We thought calcium would induce a fold instead it induced oligomerization. On the left hand we have no calcium. Expect to see a well dispersed spectrum along with at least 87 different resonances corresponding to the 87 amino acids of our peptide. The clustering of peaks in the centre along with the strong intensity of peaks tells us that we have unfolded protein. Since the same refolding conditions were used as for the CD we are led to believe that the CD data is flawed. Interestingly upon titration with calcium we noticed a disappearance of peaks. This is due to the oligomerization of our protein with the addition of calcium. The line widths resonances are directly proportional to the tumbling of the molecule. If the molecule gets too big or oligomerizes the line width becomes too broad and is lost in the baseline. Disappearance of peaks due to the increased tumbling time and as a result there is a decrease in signal. Oligomerization affects peaks Not a pH thing. pH 7.0. Anything below 7 is good for NMR since N proton would
  • Calcium Binding Properties Of Fibulin 5

    1. 1. Calcium Binding Properties of Fibulin-5 Jan Sterniczuk Dr. Smith Lab
    2. 2. The Fibulin Family Timpl R., Sasaki T., Kostka G., Chu M. (2003) Nature 4, 479-489
    3. 3. <ul><li>Notch-1 </li></ul><ul><li>Comprises 40 residues with three invariant disulfide bonds paired 1-3, 2-4 and 5-6 orientation. </li></ul><ul><li>Several anti-parallel  -sheets. </li></ul><ul><li>Calcium stabilizes the EGF into a rod-like conformation. </li></ul>The calcium binding EGF Module Hambleton S. et al . Structure (2004) 12, 2173-2183 EGF 11 EGF 12 EGF 13
    4. 4. <ul><li>Vitamin-K dependent protein S </li></ul><ul><li>Not always elongated </li></ul><ul><li>variation in structure, modular structure </li></ul><ul><li>hard to know what to expect </li></ul>The calcium binding EGF Module – unusual fold Biochemistry. 2005 Jun 21;44(24):8782-9 EGF-4 EGF-3
    5. 5. Structure and Function of Fibulin-5 RGD <ul><li>Predominantly expressed in developing arteries (Nakamura 1999) </li></ul><ul><li>Essential determinant of elastic fiber organization (Nakamura 2002) </li></ul><ul><li>Involved in smooth muscle cell proliferation and migration (Spencer 2005) </li></ul><ul><li>Interacts with lipoprotein(a) (Kapetanopoulos 2002) </li></ul>proposed Ca 2+ binding E pidermal G rowth F actor (EGF) Modules Extended C-terminal Region SP 2 3 4 5 6 1 N C
    6. 6. Fibulin-5’s cbEGFs proposed Ca 2+ binding Epidermal Growth Factor Modules Extended C-terminal Region RGD Does fibulin-5 bind Ca 2+ and if so, what is its role? -Currently, no structural or biophysical characterization studies SP 2 3 4 5 6 1 N C
    7. 7. Thesis Objectives <ul><li>To elucidate the nature of calcium binding to fibulin-5. </li></ul><ul><ul><li>Purify and refold 5 th and 6 th EGF modules with varying lengths of the C-terminal region of Fibulin-5 </li></ul></ul>
    8. 8. Protein Derivatives of Fibulin-5 448 a.a. 351 5 6 264 344 EGF-A 5 6 264 EGF-B SP 2 3 4 5 6 1 N C
    9. 9. Experimental Procedure Protein Constructs Protein Expression (Bacterial System) Refolding in redox buffer – cysteine/cystine, DTT Size-exclusion chromatography 2D NMR Circular Dichroism Ni 2+ affinity column (8M Urea, DTT) Evaluate refolding Analytical Ultra Centrifugation
    10. 10. Expression and purification of and Expression of EGF-B at 37 °C std. uninduced induced A A Ni 2+ elution B Ni 2+ elution A Concentrated B Concentrated B Size-exclusion fraction Expression of EGF-A at 37°C uninduced induced B std. 5 6 EGF-A 5 6 EGF-B 75 50 25 20 75 50 25 20
    11. 11. Purification of (Size exclusion chromatography) 1 2 3 5 6 EGF-B 20 25 std 1 2 3 15
    12. 12. CD analysis of calcium-binding to 5 6 EGF-B μ M μ M Ca 2+
    13. 13. CD analysis of 5 6 EGF-A
    14. 14. CD Spectra of other EGF-like modules Recombinant cbEGF 7-9 microneme protein 4 Periz et al, Molecular & Biochemical Parasitology 143 (2005) 192-199.
    15. 15. Experimental Procedure Protein Constructs Protein Expression (Bacterial System) Refolding in redox buffer – cysteine/cystine, DTT Size-exclusion chromatography 2D NMR Circular Dichroism Ni 2+ affinity column (8M Urea, DTT) Evaluate refolding Analytical Ultra Centrifugation
    16. 16. 15 N Labeling <ul><li>Growth of cells on minimal media </li></ul><ul><ul><li>Grow cells in salts and minimal nutrients </li></ul></ul><ul><ul><li>Force cells to incorporate 15 N </li></ul></ul><ul><ul><li>Only source of N  15 NH 4 Cl </li></ul></ul>15 N H PROBE
    17. 17. 2D 1 H- 15 N-HSQC Backbone amide nitrogen Simplistic Spectrum Polypeptide 15 N 1 H H 15 N R.f. Pulse Relaxation
    18. 18. 2D 1 H- 15 N HSQC NMR analysis of the folding and Ca 2+ -binding properties of No Ca 2+ 5 6 EGF-B 10mM Ca 2+ 5mM Ca 2+

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