Perspectives of StructureSequence Dependent Stability of Collagen and Interaction of Polyphenol Mole

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
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

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Collagen Triple Helix
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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

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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
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

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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

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Frequency of Occurrence of Selected Triplets in
Collagen
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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

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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

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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

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Model Collagen Triplets for Ab initio and DFT
calculations
Gly-Pro-Hyp Extended conformation

• Gly-Pro-Hyp collagen-like conformation

Superimposed structures of Gly-Pro-Hyp in both
conformations
18
Relative Energy of Proline Containing Triplets
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Relative Energy of Hyp Containing Triplets
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Relative Energy of Triplets without Imino acids
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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

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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

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Free Energy Solvation
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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

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Free Energy Cycle
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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

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Triplets Involved in the Stability of Collagen A
Propensity Scale
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A Propensity Scale
Collagen
a-helix
b-turn
b-sheet
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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

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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

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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

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Computer Simulation of Collagen like
Peptide-Tannin Interaction
Collagen -Catechin
Collagen -Epicatechin
Collagen Gallic Acid
Collagen -Quercetin
35
Interaction of gallic acid collagen like peptides
Gallic Acid

• 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

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• 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

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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

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• 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

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• 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

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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

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• 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

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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
43
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
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Hydrogen Bonded Complexes of Glutamic acid
Dipeptide and Gallic acid
C2
Hydrogen Bonded Complex of Serine Dipeptide and
Gallic acid
Hydrogen Bonded Complexes of Lysine Dipeptide and
Gallic acid
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-ve MESP of serine gallic complex (C1)


-ve MESP of lysine gallic complex (C2)
-ve MESP of glutamic-gallic complex (C4)
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• 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

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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-G
  lu-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

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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)

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Interaction of Polyphenolics with Collagen-like
Peptide Sequence
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Molecular Electrostatic Potential Surface (MESP)
of Gallic acidCollagen-like Peptide Complex
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Binding Energies of Polyphenol-Collagen-like
Peptide Complexes
1 Polyphenolic molecule interacted around the
serine and glutamic acid residue of the
collagen-like peptide sequence. 2 Polyphenolic
molecule interacted around the arginine residue
of the model collagen-like peptide sequence. 3
Polyphenolic molecule interacted around the
hydroxyproline residue of the model collagen-like
peptide sequence.
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Lessons from Molecular Modeling Studies

• Molecular modeling studies have provided a basis
  to identify the interaction process involved in
  tanning
• Catechin exhibited stronger binding, as compared
  to other polyphenolics chosen for the study
• Many of the complexes exhibited hydrogen bonding
  and some exhibited electrostatic and weak
  interactions
• MESP has revealed a lock and key type of
  electrostatic interactions involved in the
  stabilization of gallic acid and collagen-like
  peptide complex

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Geometrical Issues in binding small molecules by
collagen A Prospective Analysis
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Computational Details

• Four representative polyphenol molecules viz.,
  gallic acid, catechin, epigallocatechingallate
  and pentagalloylglucose chosen for interaction
  studies
• 24-mer collagen triple helix corresponding to
  residues 193 to 216 (2?1 and 1?2 chains) of the
  native Type I collagen is constructed using the
  GENCOLLAGEN package
• Following is the amino acid sequence of triple
  helix,
• Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Ala-Lys-
  Gly-Pro-Ala-Gly-Asn-Hyp-Gly-Ala-Asp-Gly-Gln-Hyp
  ?1
• Gly-Glu-Val-Gly-Leu-Hyp-Gly-Leu-Ser-Gly-Pro-Val-
  Gly-Pro-Hyp-Gly-Asn-Ala-Gly-Pro-Asn-Gly-Leu-Hyp
  ?2

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• The 24-mer triple helix and polyphenols are
  minimized using CVFF with a dielectric constant
  of 4.0
• Collagen - an inside out protein
• Side chain hydroxyl group of the amino acids,
  serine and hydroxyproline, carboxyl group of
  aspartic acid, amino group of lysine and amide
  group of aspargine are potential interacting
  sites for formation of hydrogen bonds with
  polyphenols

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Energy minimized structures of polyphenols
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Catechin
Epigallo Catechin Gallate
Vegetable Tannins
Penta galloylglucose
Gallic acid
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Energy minimized structure of 24-mer collagen
triple helix
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Complex between aspargine of T.Helix and gallic
acid
Complex between aspartic acid of T.Helix and
catechin
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Complex between lysine of T.Helix and
epigallocatechingallate
Complex between aspargine of T.Helix and
pentagalloylglucose
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Binding energies different complexes between
polyphenols and triple helix
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Hydrogen bonds of complexes their length and
angle
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Total and contact surface areas of the collagen
like triple helix and polyphenols in Å2
CSA Contact surface area TSA Total Surface
Area
Solvent inaccessible surface areas of the
complexes in Å2
AT Solvent inaccessible Total Surface Area BT
Solvent inaccessible Contact Surface Area TSA
of the complexes are in the range of 3840
4160 CSA of the complexes are in the range of
1160 1250
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Plot of interfacial interacting volume Vs Binding
energy of the complex
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Plot of effective solvent inaccessible contact
volume Vs Binding energy of the complex (inset)
Plot of effective solvent inaccessible contact
surface area Vs Binding energy of the complex
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Plot of inverse of interacting interfacial volume
(1/Int.Vol.) Vs inverse of binding energy(1/B.E)
of the complexes
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• Ligation phenomena in collagen is being
  influenced by geometric parameters
• Collagen complexation with small polyphenolic
  molecules, there may exist some minimum
  geometrical sizes and binding energies for
  influencing the long range ordering processes
• Ability of polyphenol bearing flavanoid structure
  in management of arthritis and tanning may well
  result from their ability to reduce accessibility
  of solvent(water) to molecular surfaces of
  collagen
• The present investigation offers the possibility
  to understand further recognition of phenomena
  associated with protein-protein and DNA-protein
  interactions in general, based on interfacial
  volume and surface areas

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Thank You