Although the native conformation of a protein is thermodynamically its most stable form, this stability is only marginal. As a consequence, globular proteins have a certain amount of flexibility in their backbones which allows for conformational changes in the course of their biological function. In the course of this thesis, we study protein models at the edge of stability in different contexts: (1) First, we use molecular dynamics to determine the force needed to rupture a chain molecule (an unfolded protein) being stretched at constant loading rate and temperature. When all energy bonds of the molecule are identical, we find that the force F depends on the pulling rate r and temperature T according to F ~ const -- T 1/3|ln(r/T)|1/3 When a single weak bond is introduced, this result is modified to F ~ const -- T2/3|ln(r/ T)|2/3 This scaling, which is model independent, can be used with force-spectroscopy experiment to quantitatively extract relevant microscopic parameters of biomolecules. (2) Second, we study the structural stability of models of proteins for which the selected folds are unusually stable to mutation, that is, designable. A two-dimensional hydrophobic-polar lattice model is used to determine designable folds and these folds were investigated under shear through Langevin dynamics. We find that the phase diagram of these proteins depends on their designability. In particular, highly designable folds are found to be weaker, i.e. easier to unfold, than low designable ones. This is argued to be related to protein flexibility. (3) Third, we study the mechanism of cold denaturation through constant-pressure simulations for a model of hydrophobic molecules in an explicit solvent. We find that the temperature dependence of the hydrophobic effect is the driving force for cold denaturation. The physical mechanism underlying this phenomenon is identified as the destabilization of hydrophobic contact in favor of solvent separated configurations, the same mechanism seen in pressure induced denaturation. A phenomenological explanation proposed for the mechanism is suggested as being responsible for cold denaturation in real proteins.

Globular proteins are important ingredients in many food formulations because of their nutritional value and their various functional properties such as gel formation. Proteins form gels by polymerization into a three-dimensional matrix. The rheological properties of the resulting viscoelastic solids can be obtained by Instron measurements. In the present work, gels were obtained by heating solutions of bovine serum albumin (BSA) in D$ sb2$O and egg albumin in H$ sb2$O under different conditions (i.e., pH, salt concentration, protein concentration, time of heating and temperature), and their gel strengths were measured by a compression test (Universal Testing Machine LRX). The Fourier transform infrared (FTIR) spectra of the same gel samples were recorded in order to investigate the changes in protein structure at the molecular level accompanying gel formation. Intermolecular $ beta$-sheet formation was found to increase as gel formation progresses at the expense of intramolecular $ beta$-sheet and $ alpha$-helix structures. For BSA, maximum gel strength was obtained around pH 7. The addition of salt had little effect on the gel strength while increase in protein concentration, time of heating and temperature increased the gel strength. The rate of denaturation of BSA and ovalbumin and of mixtures of these proteins in the ratios 9:1, 1:1, and 1:9 was also investigated by measuring the peak height of an aggregation band at 1618 cm$ sp{-1}$; some inhibitory effects of BSA on ovalbumin aggregation were observed. Correlations between the measured gel strengths and the amide I band profile in the FTIR spectra were examined using partial-least-squares (PLS) regression. These studies reveal that gel strengths of a particular protein gel could be adequately predicted from their infrared spectra without the need to carry out the rheological compression measurements.

Variable-temperature Fourier transform infrared spectroscopy can be employed to monitor the denaturation and aggregation of proteins during heat treatment. Information on the changes that occur in protein secondary structure upon heating is provided by detailed examination of the amide I band, as different protein conformations have characteristic amide I frequencies. The objectives of the present study were: (i) to study the changes in protein structure that occur during gelation of whey protein isolate (WPI) and (ii) to correlate the changes in protein structure observed under different physico-chemical conditions to rheological properties of WPI gels prepared under the same conditions. / The FTIR spectra of D2O solutions of WPI at different pHs, ranging from 3 to 10, were recorded as the temperature of the solution was increased from room temperature to 95°C in 5°C increments. In all the solutions studied, the formation of two new bands at 1618--1623 cm -1 and 1678--1684 cm-1 was observed upon heating; these bands are characteristic of aggregate formation and have been previously assigned to antiparallel beta-sheet structure. As the pH increased from 3 to 10, the aggregation temperature of WPI decreased from 85°C to 65°C. / The rheological properties of WPI gels were studied by employing an Instron Universal Testing Machine. The Instron measurements demonstrated that protein concentration, heating temperature, and heating and cooling time are directly related to gel strength. The changes in gel strength as a function of cooling time (for gels prepared by heating at 75°C for 45 min) were correlated to FTIR spectral data for WPI solutions subjected to the same treatment.

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Hyperbaric pressure has been shown to affect the secondary structure of whey proteins such as beta-lactoglobulin (beta-lg). There is limited research, however, regarding the optimal conditions by which pressurization of whey proteins could lead to irreversible changes in secondary structure including the reduction of intramolecular disulfide bonds. Irreversible changes in protein conformation and breakage of disulfide bonds of whey proteins induced by high pressure might result in an increase in their digestibility and a reduction of allergenicity. Hence, the overall objective was to explore the capability of hyperbaric pressure to alter irreversibly the secondary structure of whey proteins and thereby alter their allergenic and nutritional properties. The behaviour of different genetic variants of beta-lg was studied employing variable-pressure Fourier transform infrared (FTIR) spectroscopy to establish the optimum pressures needed for their denaturation. The results showed reversible effects of pressures up to 12.0 kbar on the secondary structure of three main genetic variants of beta-lg. The individual response of the genetic variants to pressure was distinguishable despite their subtle structural differences. Pressure-induced conformational changes were studied separately in bovine serum albumin, Ca++-saturated alpha-lactalbumin and Ca ++-free alpha-lactalbumin by FTIR spectroscopy. The studies revealed that the presence of Ca++ ion and the number of disulfide bonds protects the protein molecules against pressure. As whey proteins appeared to be resistant to denaturation upon single applications of high pressure up to 1200 MPa, we developed a novel pressure processing using a combination of pulse and continuous modes at lower pressures of 400 MPa which led to irreversible denaturation of whey protein structure and disulphide bond breakage. Weanling rats fed with whey protein isolates treated by this novel low pressure processing technique showed enhanced grow

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The structural and mechanical properties of a surface often play an integral part in the determination of the cell adhesion strength and design parameters for creating a biodegradable electrospun scaffold. Nanofibers composed of the globular proteins bovine serum albumin (BSA) and fibronectin were produced by electrospinning with the electrospun protein scaffold serving as an extracellular matrix to which adhesion interaction will exist with cells via cell surface integrin. This interaction is vital in regulation cell differentiation, growth and migration and cell adhesion. We will demonstrate the ability to manipulate ligand-receptor interaction, the properties of the electrospun fibers, control and the formation of focal adhesions sites in cells cultured on the fibers with the ultimate goal of developing a biomimetric scaffold to investigate how cell adhesion molecules modulate cell behavior in a 3-dimentional culture.

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