Kimberly M. Taylor

Code for collagen's stability deciphered

The most abundant protein in animals is collagen. In connective tissue, this protein is present as chains wound in tight triple helices which are organized into fibrils of great tensile strength and thermal stability,. We propose a new explanation for this stability.

A hyperstable collagen mimic

BACKGROUND Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies.

Broadband 10-300 GHz stimulus-response sensing for chemical and biological entities.

By illuminating the sample with a broadband 10-300 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, however, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimetre-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in single-wavelength microwave reflections coincide with accepted visible absorption spectra, pointing the way towards gaining quantitative chemical and biological spectra from broadband terahertz systems.

Ultra-sensitive detection of protein thermal unfolding and refolding using near-zone microwaves

We use planar slot antennas proximal to proteins in solution to detect changes in conformation. The antennas are attached to fused-quartz or glass sample holders and the cuvette/antenna assembly is placed in the sample holder of an optical spectrophotomer (either UV/VIS or fluorescence polarization), allowing simultaneous dielectric and optical measurements. Return loss is recorded using a vector network analyzer. This system was used to study the equilibrium thermal unfolding and refolding of a small globular protein, as well as the binding of small hormones to a receptor. Good agreement between optical and microwave measurements was obtained for all systems studied. We show that microwave measurements can be made at protein concentrations as low as 0.3 ng/mL (19 pM), several orders of magnitude lower than that required for optical spectroscopy. The results from these experiments demonstrate that resonant slot antennas can be used to detect changes in protein conformation and the presence of microwave radiation does not perturb the system under study.

SPECTROSCOPY WITH ELECTRONIC TERAHERTZ TECHNIQUES FOR CHEMICAL AND BIOLOGICAL SENSING

By illuminating the sample with a broadband 10-500 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimeter-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in microwave reflections coincide with accepted visible absorption spectra, pointing the way toward gaining quantitative chemical and biological spectra from broadband terahertz systems.

The stereoelectronic basis of collagen stability

We conclude that collagen stability does not rely on bridging water molecules. Rather, stereoelectronic effects preorganize collagen strands. Specifically, an electronegative substituent in the 4(R) position of a proline residue favors a trans peptide bond, as is necessary for triple helix formation.

Ultra-sensitive microwave detection of protein conformational changes

A coaxial driven planar slot antenna resonant in the microwave regime was used to detect changes in protein conformation induced by temperature modulation. Simultaneous dielectric and UV/VIS spectroscopy measurements were obtained by attaching the slot antenna to a fused-quartz UV/VIS sample holder. Similar thermodynamic parameters (midpoint temperature and enthalpy) were obtained from both techniques over a wide concentration range and under a variety of pH condition. Here we demonstrate that a near-field antenna can be used to detect changes in conformation at very low protein concentrations, and that the protein is not destabilized by the presence of microwave power.

Modulating the conformational stability of triple-helical collagen by chemical modification

The conformational stability of triple helical (ProHypGly)10 can be altered by chemical modification of the Hyp hydroxyl group.