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Temperatue Dependancy of Collagen-CMP integration

Collagen is one of the most important structural proteins in the body and has the potential to become an important tool in medicine. A novel method of modifying collagen is to use Collagen Mimetic Peptides (CMPs), amino acid sequence (Pro-X-Gly)n.

ingrid spielman

on 5 May 2010

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Transcript of Temperatue Dependancy of Collagen-CMP integration

Temperature Dependancy of Collagen-CMP Binding Collagen is one of the most important structural proteins in the body and has the potential to become an important tool in medicine. A novel method of modifying collagen is to use Collagen Mimetic Peptides (CMPs), amino acid sequence (Pro-X-Gly)n, which bind to lose segments of collagen to form a triple helix. These CMPs can be modified with antibiotics, cell adhesion peptides, growth factors, or medications, which all can be released into the body at controlled rates. Previous studies have shown that CMPs will adhere to collagen but the experiment did not fully control for CMP temperature and collagen denaturation. My research explores the temperature dependency of the Collagen-CMP association Collagen 26% by mass protein in body gives structure and strength
Hydration Shell
Side Chain Interactions
Interchain Hydrogen Bonding Ingrid Spielman Michael Yu Lab
May 5th, 2010 Background Collagen Mimetic Peptides Triple Helix Formation Previous Work Problem With Set up Collagen Research Make Peptide Make and Denature Films Refolding kinetics Solution Design Future References Questions? acknowledgement Future Work Discussion Facile modification of collagen directed by collagen mimetic peptides. Wang AY, Mo X, Chen CS, Yu SM.J Am Chem Soc. 2005 Mar 30;127(12):4130-1

Spatio-temporal modification of collagen scaffolds mediated by triple helical propensity. Wang AY, Foss CA, Leong S, Mo X, Pomper MG, Yu SM.Biomacromolecules. 2008 Jul;9(7):1755-63. Epub 2008 Jun 12

Collagen Primer in Structure, Processing and Assembly. Jürgen Brinckmann, Holger Notbohm, P. K.Müller. Springer. 2004.

Self-association of collagen triple helic peptides into higher order structures. Kar K, Amin P, Bryan MA, Persikov AV, Mohs A, Wang YH, Brodsky B. J Biol Chem. 2006 Nov 3;281(44):33283-90. Epub 2006 Sep 8.
Michael Yu
For intellectual discussions and training:
Yang lee, Tania Chan, Patrick Stahl, Dan Kim
1. Continue modifying/optomizing CMP-collagen film binding study and confirm results with CF-G3-CMP6 and RCMP

2. Confirm CMP- collagen binding via alternate method to film binding studies

3. Use Matrix metalloproteinases (MMP) to cut the collagen films at very specific points. These cuts will create more open ended collagen strands which may induce higher collagen mimetic peptide binding. 48-well plate with CMP (yellow) added to collagen films
Experimentation Collagen Gelatin Heat "We hypothesized that the propensity of CMPs to form collagenlike triple helices may enable it to bind to partially denatured collagen by associating with disentangled domains of the collagen molecules." Glycine: 57 g/mol
Hydroxyproline: 97 g/mol
Proline: 114 g/mol
Total: 268 g/mol
X 9 = 2412 g/mol
+ 3 glycine: 171 g/mol
+ CF: 376.32 g/mol
Total: 2960 g/mol
+ salt: ~ 15 g/mol
PEAK: 2975 g/mol (MALDI) Matrix-assisted laser desorption/ionization
Mass Spectrometry 2975 g/mol Intensity Molecular weight (g/mol) Triple Helix Content Thermally denatured collagen I:
Quenched at 4°C regains triple helix optical properties
However, arrangement is no longer the same
Chains associate into misaligned structures characteristic of gelatin

In the body:
Body temperature is just below the melting temperature of a collagen triple helix
higher order structure increases stability
Slowely unravel and enzymatically degraded via MMP (matrix metalloproteases) Glycine
Hydroxyproline 15-30 aa long Easily conjugated to bioactive components
Free C and N terminus for covalent condensation-like reaction
Will form a triple helix with itself below Tm
Helix Single Strand k1 3 levels of heirarchy 1. twists on itself
2. right handed triple helix
Gly in center
3. Fiber bundles Concentration of CMP uM = 0.032 (UV-vis reading) + 0.052
R² = 0.997 Circular dicroism Z = # strands connected to form triple helix When a solution of collagen I or another fibril-forming collagen without disulfide bonds is denatured thermally and then quenched at 4°C, recovery of the optical properties of the triple helix such as optical rotation or ellipticity is relatively fast and nearly complete. The solutions, however, do not recover the viscosity of a solution of the native protein because the chains have associated into a variety of misaligned structures characteristic of gelatin.
Add hot solution of CF tagged peptide to film
Incubate, wash, use UV-vis to determine concentration of remaining CMP

CMP 10 Binds above its melting Temp (75C)
CMP 7 and 6 also bind above their melting temperature
CMP 7 and 6 do not bind as well at 80C
CMP random control does not bind well Applications Collagen, as an important ECM scaffolding can be used by engineers to create artificial scaffolding for cell growth and differentiation. It is abundant in nature and is easily extracted from animals.
corneal shields
tissue sealants
skin replacement
damaged tissue support
blood vessels Interactions However normal ECM also contains laminin, fibronectin, vitronectin, fibrinogen, glycosaminoglycans, hyaluronic acid, etc… which are all important for aiding in cell adherence and differentiation. We need a way to add these components to collagen matrixes without having them diffuse away on a relatively short time scale.
GAGs, HA Recruit Cells QK (VEGF derived)
RGD (fibronectin)
YIGSR (laminin) cell adherence and differentiation k2 k1 ~= k2 Glycine
Hydroxyproline Visualize by tagging with Carboxyfluorescein (CF) x3 Is it the collagen denaturation what allows for CMP binding or is it the energy added to the system?
Paper 1 images of film surface Paper 2 UV-vis from sample wash
Decreased volume The collagen locally may denature with the addition of a Collagen mimetic peptide (CMP) hot solution Denature Films before CMP addition We know that CMP triple helix does not reform instantly Cool the CMP peptides quickly before adding them to the film Denature the films before performing the binding study C - control (cold CMP or RCMP)
Q - quenched CMP 1. Heat and Quench
100 uM
200 uM
400 uM
2. Measure ellipticity
at 225nm over time
When heated Collagen single strands no longer exhibit triple helix right handedness Ellipticity peak found at 225nm Time of incubation
Concentration of CMP
Denaturation of film Problems:
1. Film comes off plate
2. UV-vis readings unstable
3. Difficult to wash films Hypothesis 1: Quenched CMPs will not bind as well to collagen as Hot CMPs

Hypothesis 2: CMPs may bind more readily to denatured film (gelatin) than undenatured film due to an increase in unravelled collagen segments
However, low probability of CMPs binding to single strands of collagen The use of MMP is particularly interesting because MMP is an enzyme secreted by tumors to the surrounding area in order to make room for its growing mass. It would be interesting to see if CMP will bind more greatly to these regions and we may be able to use the CMP as an early cancer detection system Preliminary Results Wang AY. Biomacromolecules. 2008 CF-G3-CMP9 Completely denatured films do not retain CMPs
Undenatured films retain both heated and quenched CMP ECM Components
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