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V. Subramanian Chemical Laboratory Central Leather Research Institute Chennai

Perspectives of Structure-Sequence Dependent Stability of Collagen and Interaction of Polyphenol Molecules with Collagen. V. Subramanian Chemical Laboratory Central Leather Research Institute Chennai. Introduction.

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V. Subramanian Chemical Laboratory Central Leather Research Institute Chennai

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  1. Perspectives of Structure-Sequence Dependent Stability of Collagen and Interaction of Polyphenol Molecules with Collagen V. Subramanian Chemical Laboratory Central Leather Research Institute Chennai

  2. Introduction • Collagen is an extremely important protein, which provides mechanical strength and structural integrity to connective tissues • Nineteen different collagen types identified till date • The identifying motif of the collagen is triple helix • Prof Ramachandran and co-workers and Rich and Crick • The Gly-X-Y is the general repeating sequence of the collagen (33% of Gly) • Mutations in collagen chain can render the fibrils unstable

  3. Triple Helix • The collagen triple helix constitutes the major motif in fibril forming collagen and also occur as a domain in non-fibrillar collagens • Hydrogen bonding and presence of high content of imino acids provide stability to the three dimensional structure of collagen • The role of water mediated hydrogen bonding and hydration also play a crucial role in the stability of collagen • Recent experimental studies revealed that the presence of Arg in the Y position provides equal stability when compared to Gly-Pro-Hyp • The destabilizing nature of Asp in the Y position is also evident from the experimental studies • Therefore assessment of propensity of various amino acids to form collagen like peptides is an important area of research activity

  4. Stabilizing Inter-strand H-bonds Collagen Structure: An Indian Origin • Single Vs two hydrogen bond(s) • If X and Y positions are imino acids, there is no possibility of forming two hydrogen bonds • The incorporation of other amino acids provides a possibility of readdressing this question

  5. Collagen Triple Helix

  6. Propensity of Various Amino Acids to Form Collagen Like Motif: Guest Host Approach • Propensity of various amino acids to form alpha helix and beta sheet have been addressed • Host-Guest peptide approach has been used to estimate the propensity • The presence of various amino acids not only influences the three dimensional structure but also the stability of collagen • The amino acid propensity-stability-function is an important area of research in molecular biophysics • Several experimental studies have been carried out on model collagen-like peptides to establish the propensity of various amino acids to form collagen

  7. Triple Helix Propensity Scale • Circular dichrosim Spectroscopy has been used to develop triple helix propensity of various amino acids • The molar elipticity was monitored at 225nm while sample temperature was increased from 0 to 800 C • The melting curves were used to calculate fraction of folded states • Fraction folded has been used to compute vant Hoff enthalpies and free energy • These information provides experimental basis for predicting relative stabilities of various amino acids to form collagen like structure • Propensity scales will be used to compare the results obtained from modeling and simulations

  8. Thermodynamics parameters for the Guest Host peptides Biochemistry, 1996, 32, 10262. Unraveling the Stability of Collagen: Experimental Studies by Brodsky and Co-workers • Host-Guest approach has been used to introduce new sequences in the general repeating Gly-Pro-Hyp sequences • Parameters such as • melting temperature • thermodynamics parameters from melting studies • G of stabilization of the host-guest collagen-like peptides have been studied to identify the influence of amino-acids towards the stability of collagen

  9. Propensity data from Brodsky work Melting Temperature & Thermodynamic parameters for the Guest Host peptides in Y position Melting Temperature & Thermodynamic parameters for the Guest Host peptides in X position Biochemistry, 2000, 39, 14960.

  10. Collagen in Diseases • Mutation in collagen genes COL1A1 and COL1A2 leads to Osteogenesis Imperfecta (OI), a brittle bone disease • A point mutation in one of types collagen genes can cause disease • One of the main cause for OI is GlyAla mutation • Glycine substitutions to another amino acid more severe than mutations of X or Y in Gly - X - Y triplet. • Understanding the stability of collagen upon mutation becomes necessary • Since collagen is a large protein, it is difficult to study the influence of amino acids • Various attempts have been made to probe the effect of mutation in model collagen-like peptide sequences

  11. Collagen in Diseases • Mutations in collagen leads to Osteogenesis Imperfecta (Type –I), Chondrodysplasis (type II), Ehlers-Danlos syndrome (type III), Alport syndrome (type IV), Bethlem myopathy (type VI) etc • Mutation in collagen genes COL1A1 and COL1A2 leads to Osteogenesis Imperfecta (OI), a brittle bone disease • A point mutation in one of type I collagen genes can cause disease • One of the main causes for OI is GlyAla mutation • Glycine substitutions to another amino acid is more severe than mutations of X or Y in Gly - X - Y triplet Understanding the stability of collagen upon mutation becomes necessary

  12. Collagen mimics and Biomaterial applications • Various physical and chemical properties make collagen as a versatile material for biomaterial applications • Studies on Collagen mimetics have been made to understand the strength of triple helix and for their application in biomaterials • In collagen mimetics, a variety of unnatural amino acids are incorporated in X and Y positions • K. N. Ganesh* and his coworkers have used 4 amino proline containing collagen like sequences • Murray Goodmann$ and his group made an attempt to template assembling of collagen like peptides using conformationally constrained organic molecule *JACS, 1996, 118, 5156 $JACS, 2001, 123, 2079

  13. Frequency of Occurrence of Selected Triplets in Collagen

  14. Propensity of Various Amino Acids to Form Collagen Like Motif • The propensity of various amino acids to form alpha helix and beta sheet have already been established • Host-Guest peptide approach has been used to estimate the propensity • The presence of various amino acids not only influences the three dimensional structure but also the stability of collagen • The amino acid propensity-stability-function is an important area of research in molecular biophysics • Several experimental studies have been carried out on model collagen-like peptides to establish the propensity of various amino acids to form collagen

  15. Issues addressed • To determine the stability of collagen upon substitution of Gly-Pro-Hyp by other collagen-like triplets • To develop the propensity scale for various amino acids to form collagen-like peptides based on free energy of mutation • To probe the interaction between model collagen like peptides with polyphenols

  16. Methodology • Ab initio and DFT calculations have been performed on collagen like triplets in both collagen and extended conformation • Free energy of solvation for these triplets have been computed for both conformations using Polarizable Continuum Method • Free energy of solvation has been used to compute the stability and amino acid propensity • The stability of these peptides have also been analysed by calculation of hardness • Free energy of various triplets have also been computed using classical molecular dynamics simulations • Using these values free energy change has been quantified

  17. Model Collagen Triplets for Ab initio and DFT calculations Gly-Pro-Hyp collagen-like conformation Gly-Pro-Hyp Extended conformation Superimposed structures of Gly-Pro-Hyp in both conformations

  18. Relative Energy of Proline Containing Triplets

  19. Relative Energy of Hyp Containing Triplets

  20. Relative Energy of Triplets without Imino acids

  21. Important Observations • The triplets containing proline or hydroxy proline are more stable in collagen-like conformation • Proline sterically restricts the N-Crotation and it has limited values of , – 63 ±15 degrees • Hence, proline can not be found in other known major protein motif • The dihedral angle corresponding to conformational energy minima for proline has been found to be –75 and 145o (, ) • It can stabilize secondary structure of protein only when the allowed values of all other amino acids coincide with that of proline

  22. Important Observations ………Contnd. • It is evident from the relative energy that Gly-Gly-Hyp does not stable in collagen like conformation • Recent experimental evidence confirms that glycine in the second position destabilizes the collagen triple helix • Solvation drastically alters the relative energy • Proper ordering of the stability of various triplets needs geometry optimization in solvent media

  23. Free Energy Solvation

  24. Important Observations • Solvation free energy of collagen-like sequences indicates that the triplets in collagen-like conformation can be hydrated better than its extended counterpart • The presence of polar and non-polar residues in the sequence drastically influences the solvation • Specifically Arg either in second or third position influences the solvation • Arg stabilizes the collagen-like sequence similar to stability provided by Hyp in the Y position

  25. 1 2 3 4 Free Energy Cycle

  26. Assessment of Stability Using G • The propensity to form collagen-like sequence has been calculated using Gly-Pro-Hyp as reference • The calculated G ranges from 0.0 to 15.8 kcal/mol • The change in the free energy of Gly-Pro-Pro and Gly-Pro-Flp is close to Gly-Pro-Hyp • The most stable sequence is Gly-Pro-Hyp • The general trend correlates well with the experimental values derived from melting temperature studies on model systems

  27. Triplets Involved in the Stability of Collagen: A Propensity Scale

  28. A Propensity Scale Collagen a-helix b-sheet b-turn

  29. Chemical Hardness • Global hardness of various triplets in collagen-like and extended conformation has been calculated • It interesting to note that the chemical hardness values are more for the triplets in collagen-like conformation than extended conformation • Experimentally, Asp in the triplet does not favor collagen folding • Chemical hardness for Gly-Pro-Asp is observed to be less compared to the other sequences

  30. Important Observations • B3LYP/6-31 G* level of theory predicted that collagen-like conformation of the Gly-Pro-Hyp is stable than the extended conformation by 0.46 kJ/mol • Hardness of triplets of sequences Gly-X-Y (without Hyp and Pro) is lower than the triplets containing Pro and Hyp residues

  31. Emerging Roles of Computational Techniques in Tanning Theory • Computer model of bovine type I collagen has been simulated • Early report of molecular modeling of tanning processes has been made • Model peptides for collagen has been selected and interactions with various tanning materials simulated using force field as well as Density Functional Theoretical methods • Binding energies for various interactions of collagen like peptide with tannin molecules have been estimated

  32. Computer Simulation of Collagen –like Peptide-Tannin Interaction Collagen -Catechin Collagen -Epicatechin Collagen –Gallic Acid Collagen -Quercetin

  33. Interaction of gallic acid collagen like peptides • Gallic acid is a good anti-oxidant present in many plant sources • Gallic acid has been shown to selectively induce cell death in cancerous cell lines by binding to specific receptors or enzymes • Gallic acid finds major role in stabilization of collagen in tanning process of leather making • Collagen is an important and abundant connective tissue protein in animal kingdom Gallic Acid

  34. Collagen assemblies are stabilized by covalent and non-covalent interactions • A fundamental understanding on the interaction of gallic acid with collagen is important to unravel the nature of interactions that are required for the stabilization of collagen matrix • Theoretical calculations can be used for the determination and quantification of such interactions • In this view present investigation focuses on determining the interactions of different dipeptides with gallic acid • Such a study can not only be correlated to stabilization process involved in collagen but also will lead to the advancement on the knowledge of peptide-ligand interaction

  35. Computational details • Three classical dipeptides of amino acids glutamic acid, lysine and serine chosen for the interaction studies with gallic acid • Dipeptides imposed with the  and  corresponding to the angles of collagen • Dipeptides and gallic acid built and energy minimized using modules available in Insight II(MSI, USA) • Four functional sites namely 3 OH groups and one COOH group present in the gallic acid identified to have the potential to interact with the side chain groups of the dipeptide • The geometry of the complexes optimized by a semi-empirical PM3 method using Gaussian98 suite of programs

  36. Energy of the complex calculated using both Hartree Fock (HF) and DFT based B3LYP methods using 3-21G* & 6-31G* basis sets employing Gaussian 98w suite of programs • The interaction energy (VINT) calculated using supermolecule approach VINT = TEcomplex – [ TEdipeptide + TEgallic acid] Binding Energy (VBE) is, VBE = - VINT

  37. Molecular electrostatic potentials (MESP) are useful in understanding the weak and non-covalent interactions. The electrostatic potential V(r) is defined as ZA is the charge on nucleus A located at RA, and (r') is the electron density at a point r • MESP features of peptide-gallic acid complex have been studied by BLYP/DN using DMOL implemented in Cerius2 • Molecular Dynamic (MD) calculations have been done for one of the complexes, to see the time evolution of the hydrogen-bonded complex. A time step of 1fs has been chosen and the MD simulations have been performed for 600 ps including an equilibration period of 100 ps

  38. Discussion • The functional groups para-OH, two meta-OH and COOH of gallic acid have been assumed to act as a hydrogen bond donor/acceptor for different side chain groups of amino acids in dipeptide • Most of the complexes have exhibited hydrogen bonding in the complexes consisting of dipeptides-gallic acid • Complexes have exhibited binding energies in the range of 4 – 18 kcal/mol • Complexes of glutamic acid dipeptide with gallic acid have all exhibited hydrogen bonding with high binding energies

  39. Some of the complexes of gallic acid with serine and lysine dipeptide have also exhibited hydrogen bonding • The interaction with COOH group of gallic and side chain COOH group of glutamic acid exhibited the maximum binding energy • All complexes calculated by HF methods predicted lower binding energies when compared to the binding energies predicted from DFT methods • Molecular electrostatic potential estimation of various complexes provided clues on the involvement of the electrostatics involved in the interaction process

  40. Dipeptide Binding energies of gallic acid – dipeptide complex (kcal/mol) Interaction energies of different sites of gallic acid with different dipeptides calculated using Density Functional Theory (B3LYP) with basis set 3-21G* and 6-31G* m1 – OH (C1) p – OH (C2) m2 – OH (C3) COOH (C4) 3-21G* 6-31G* 3-21G* 6-31G* 3-21G* 6-31G* 3-21G* 6- 31G* Glutamic Acid 10.29 8.08 7.06 5.06 11.19 8.98 18.18 15.1 Lysine 5.51 3.75 11.65 9.51 8.97 6.53 14.51 12.18 Serine 7.7 6.32 10.96 10 6.23 5.1 12.92 10.19

  41. Dipeptide Binding energies of gallic acid dipeptide complex (kcal/mol) m1 – OH (C1) p – OH (C2) m2 – OH (C3) COOH (C4) 3-21G* 6-31G* 3-21G* 6-31G* 3-21G* 6-31G* 3-21G* 6-31G* Glutamic Acid 8.67 6.0 6.12 3.85 9.3 6.7 16.86 13.39 Lysine 3.55 –3.25 11.57 9.15 7.95 5.22 12.6 10.33 Serine 7.28 5.91 10.65 9.34 6.02 4.39 12.59 9.46 Interaction energies of different sites of gallic acid with different dipeptides calculated using Hartree Fock (HF) method with basis set 3-21G* and 6-31G*

  42. C2 C3 C4 C2 C4 Hydrogen Bonded Complexes of Glutamic acid Dipeptide and Gallic acid C2 Hydrogen Bonded Complexes of Lysine Dipeptide and Gallic acid Hydrogen Bonded Complex of Serine Dipeptide and Gallic acid

  43. -ve MESP of serine gallic complex (C1) -ve MESP of lysine gallic complex (C2) -ve MESP of glutamic-gallic complex (C4)

  44. The functional groups para-OH, two meta-OH and COOH of gallic acid have been assumed to act as a hydrogen bond donor/acceptor for different side chain groups of amino acids in dipeptide • Most of the complexes have exhibited hydrogen bonding in the complexes consisting of dipeptides-gallic acid • Complexes have exhibited binding energies in the range of 4 – 18 kcal/mol • Complexes of glutamic acid dipeptide with gallic acid have all exhibited hydrogen bonding with high binding energies

  45. Collagen-like Peptide Sequence • Difficult to handle large systems like collagen molecule for molecular simulation calculations • Interaction studies can be carried by building collagen like peptide sequence maintaining the uniqueness of collagen • A 9-mer sequence Ace-Gly-Pro-Hyp-Gly-Ala-Ser-Gly-Glu-Arg-Nme is built based on the repeatability of the sequences and on the presence of amino acids in the actual collagen molecule by imposing  and  constraints based on G N Ramachandran plot

  46. Interaction of Polyphenolics with Collagen-like Peptide Sequence • Peptide sequence and polyphenolic molecules minimized using CVFF(Consistence Valence Force Field) • The polyphenolic molecules placed near the different sites of the peptide sequence and minimized • The binding energy of the molecules with the peptide sequence calculated based on the equation, EB - Binding Energy (kcal/mol) Epolyphenolics=Total energy of the minimized structure of polyphenolic molecules (kcal/mol) Esequence = Total energy of the minimized structure of collagen-like peptide sequence (kcal/mol)

  47. Hydrogen bonded complex of Gallic acid with Serine residue of collagen like peptide (Binding Energy = 8 kcal/mol ) Complex of Quercetin with collagen like peptide (Binding Energy = 12 kcal/mol ) Hydrogen bonded Complex of Catechin with Peptide (Binding Energy = 18 kcal/mol ) Interaction of Polyphenolics with Collagen-like Peptide Sequence

  48. Positive electrostatic potential (0.7) surface of the complex of gallic acid with Glutamic acid residue of the peptide sequence Negative electrostatic potential (-0.01) surface of the complex of gallic acid with Glutamic acid residue of the peptide sequence Molecular Electrostatic Potential Surface (MESP) of Gallic acid–Collagen-like Peptide Complex

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