Organic Pedagogical Electronic Network

Stereoelectronic Effects on Collagen

Triple Helix StabilityAlexandra ZudovaSamuel Broadbent

University of Utah, 2013

Collagen Triple Helix Stability

Stereoelectronic Effects on Collagen Stability:  The Dichotomy of 4-Fluoroproline DiastereomersJonathan A. Hodges† and  Ronald T. Raines*,†,§Journal of the American Chemical Society 2003 125 (31), 9262-9263

Overview: Collagen is made up of the repeating pattern Glycine-X-Y, where X and Y are commonly L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp), respectively. By substituting X and Y with a fluorine probe, stereoelectronic effects can be observed and compared to the effects of hydrogen bonding which has been predicted to be the main contributor to the collagen triple helix strength.

This data shows that the collagen triple helix is more stable with an inductive effect due to fluorine than the predicted hydrogen bonding from the hydroxyl group being added.

Collagen Triple Helix Stability

A hyperstable collagen mimic, Steven K Holmgren*, Lynn E Bretscher, Kimberly M Taylor and Ronald T Raines, Chemistry & Biology, February 1999, 6:63-70

Effect of temperature on the conformational stability of Pro-Flp-Gly (black circles), Pro-Hyp-Gly (white circles), and Pro-Pro-Gly (white squares) peptides. Showing that the stereoelectronic effect provides a stronger triple helix that requires higher temperatures in order to denature.

Two diastereomers exist for the fluorine tagged protein. As seen in previous listed results, flp is greatly stabilizing of the triple helix in the Yaa position, whereas Flp is greatly stabilizing in the Xaa position. This is due to the gauche effect which alters the pyrrolidine ring pucker.

Applications of Collagen Triple Helix Stability- Due to the fact that collagen is the most abundant protein in animals, understanding the

basis of collagen triple helix strength can lead to the development of novel biomaterials. Collagen is often used as a tissue engineering scaffold, and its structure can be used to conjugate growth factors, drugs or other bioactive moieties

- Basement membrane collagens have complex interactions requiring more finely-tuned conformational stability, and their amino acid composition facilitates this with a less stable helix than that of fibrillar collagens

- Point mutations in collagen genes can lead to improperly folded and unstable triple helices, due to disrupted noncovalent interactions between incompatible amino acids. This can have severe pathological consequences like brittle bone disease.

- Stereoelectronic effects in the stability of the collagen triple helix have been exploited in the design of collagen mimetic peptides that selectively hybridize with denatured collagen strands in diseased tissues. These peptides have been used to target tumors and diseased cartilage to deliver imaging agents and could be used to deliver drugs.

Chapter 2, "structure, stability and folding of the collagen triple helix", of book "Collagen, Topics in current chemistry“ J. Engel · H. P. BächingerTargeting collagen strands by photo-triggered triple-helix hybridization, PNAS 2012; 109, 37 (14767-14772).Effect of 3-Hydroxyproline Residues on Collagen Stability. Jenkins, Bretscher, Guzei, and Ronald Raines. JACS 2003; 125, 6422-6427.Stereoelectronic Effects on Collagen Stability:  The Dichotomy of 4-Fluoroproline Diastereomers Jonathan A. Hodges† and  Ronald T. Raines*,†,§Journal of the American Chemical Society 2003 125 (31), 9262-9263

Problems

A hyperstable collagen mimic, Steven K Holmgren*, Lynn E Bretscher, Kimberly M Taylor and Ronald T Raines , Chemistry & Biology, February 1999, 6:63-70

Problem 1. Consider the following thermodynamic data for three collagen mimetic peptides at the triple helix-coil transition. Explain the effect of amino acids at the Y-position on Tm and ∆∆Gm, and suggest a reason for the effect of solvent on triple helix stability. Calculate the average contribution of each proline substituent on overall peptide stability for both Hyp and Flp peptides in 50 mM acetic acid. Explain the signs of the enthalpic and entropic contributions to ∆∆Gm.

Problems

Effect of 3-Hydroxyproline Residues on Collagen Stability. Jenkins, Bretscher, Guzei, and Ronald Raines. JACS 2003; 125, 6422-6427.

Problem 2. Given the following pKa data of the X component, predict the relative stability of the three collagens containing each amino acid in the X position. Also explain why the pKa of Pro-OH is highest.

Would this prediction hold for the Y position? What assumptions have you made and are they still valid? Why or why not?

X-OH group pKa of carboxyl group X-OH

3-Hyp-OH 1.62

4-Hyp-OH 1.80-1.82

Pro-OH 1.95

Solutions

A hyperstable collagen mimic, Steven K Holmgren*, Lynn E Bretscher, Kimberly M Taylor and Ronald T Raines, Chemistry & Biology, February 1999, 6:63-70

Solution to problem 1: The inductive effect of electronegative atom substitution on the proline ring stabilizes an exo ring pucker conformation in the Y position (guache effect) which stabilizes the triple helix, giving a higher Tm and ∆∆Gm for the Hyp- and Flp- substituted peptides. Because F is more electronegative that OH, the Flp peptide is more stable than the Hyp peptide. Each OH group of the Hyp peptide contributes ∆∆Gm = 0.2 kcal/mol while each F atom of the Flp peptide contributes ∆∆Gm = 0.4 kcal/mol.

In the higher ionic strength solvent PBS, these stereoelectronic effects are less significant than in acetic acid/water. The interactions between peptide strands are therefore stronger relative to the interactions with the surrounding solvent when acetic acid is used, leading to slightly higher Tm and ∆∆Gm than in PBS, on average.

Because the triple helix coil transition is a dissociation of three peptide strands, this results in a large increase in entropy due to greater degrees of rotational freedom, so the entropic change is large and positive. Since ∆∆Gm overall is positive, the enthalpic term must be large and positive enough to overcome the favorable entropy change (-T∆S) upon dissociation. From the high Tm’s shown in table 1, we know that the triple helix is very thermally stable.

Solutions

Effect of 3-Hydroxyproline Residues on Collagen Stability. Jenkins, Bretscher, Guzei, and Ronald Raines. JACS 2003; 125, 6422-6427.

Solution to problem 2. In the collagen triple helix, the carboxyl pKa of X-OH is a measure of the amino acid’s ability to accept a hydrogen bond from the glycine N-H of a neighboring strand. The strength of a hydrogen bond increases with the acidity of the donor and with the basicity of the acceptor. Since the carboxyl group is the H-bond acceptor in this case, it is acting as a base so we look for an increasing trend in pKa to predict higher helix stability.

Thus the order of stability according to H-bond strengths from least to greatest is: 3-Hyp, 4-Hyp, Pro. Pro has the highest pKa because it lacks the electronegative OH of Hyp, and thus the inductive effect that makes the carboxyl more acidic.

This analysis assumes that H-bond strength is the major factor in stabilizing the collagen triple helix, however we know that the Y position is often populated by 4-Hyp rather than Pro. To explain this, stereoelectronic effects are invoked. The inductive effect of OH in 4-Hyp stabilizes an exo ring conformation which is favored in the collagen triple helix. By contrast, the X position favors an endo ring conformation which Pro readily adopts in addition to providing a stronger interstrand H-bond. For the Y position, this preference for exo pyrrolidine rings is more significant than the destabilizing effect of weaker hydrogen bonds.

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Contributed by: Alexandra Zudova, Samuel Broadbent (Undergraduates)

University of Utah, 2013