Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm-1 in infrared spectra of dried protein-sugar samples was examined. For samples in which a high level of native protein structure was retained in the dried solid, the apparent moisture content predicted by the carboxylate band area was greater than the actual moisture content, indicating that protection results from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural protection conferred by sucrose and trehalose apparent in second derivative, amide I infrared spectra, correlates with the extent of hydrogen bonding between sugar and protein. The failure of dextran to inhibit dehydration-induced lysozyme unfolding is shown to result from the inability of the polymer to hydrogen bond adequately to the protein. Therefore, formation of an amorphous phase alone is not sufficient to maintain protein structure during dehydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryoprotectant. However, the addition of polyethylene glycol, which is known to protect proteins during freezing, but not drying, to glucose protected lysozyme structure during lyophilization. Together, these results show that hydrogen bonding between carbohydrate and protein is necessary to prevent dehydration-induced protein damage. However, hydrogen bonding alone is not sufficient to protect proteins during lyophilization in the absence of adequate freezing protection.     

The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.     

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Peptide scanning-based identification of regions of gamma-II crystallin involved in thermal aggregation: evidence of the involvement of structurally analogous,helix-containing loops from the two double Greek key domains of the molecule

Gamma crystallin is one of three structural proteins present in great abundance in the fiber cells of the vertebrate eye lens. The protein displays a tendency to aggregate readily in the course of heating, cooling, being exposed to ultraviolet radiation, or rapid refolding. To investigate the molecular mechanisms underlying such aggregation, we have employed a peptide-scanning approach aimed at identifying regions of bovine gamma-II crystallin that may be involved in intermolecular interactions leading to aggregation, using assays that measure the competitive inhibition of such aggregation by reagents drawn from a group of contiguous (overlapping) peptides derived from the sequence of the protein itself. Our results suggest that two regions, comprising residues 61-74, and 145-159, play key roles in aggregative interactions. Intriguingly, the two regions (each containing a solvent-exposed, single-turn helix in the native structure) are located in structurally analogous positions in the two homologous double Greek key (beta sheet) domains of the protein, suggesting that helix-strand conversions may operate to facilitate intermolecular beta sheet interactions during aggregation.     

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.     

Co-translational domain folding as the structural basis for the rapid de novo folding of firefly luciferase.

The 62 kDa protein firefly luciferase folds very rapidly upon translation on eukaryotic ribosomes. In contrast, the chaperone-mediated refolding of chemically denatured luciferase occurs with significantly slower kinetics. Here we investigate the structural basis for this difference in folding kinetics. We find that an N-terminal domain of luciferase (residues 1-190) folds co-translationally, followed by rapid formation of native protein upon release of the full-length polypeptide from the ribosome. In contrast sequential domain formation is not observed during in vitro refolding. Discrete unfolding steps, corresponding to domain unfolding, are however observed when the native protein is exposed to increasing concentrations of denaturant. Thus, the co-translational folding reaction bears more similarities to the unfolding reaction than to refolding from denaturant. We propose that co-translational domain formation avoids intramolecular misfolding and may be critical in the folding of multidomain proteins.     

Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin

Extensive measurements and analysis of thermodynamic stability and kinetics of urea-induced unfolding and folding of hisactophilin are reported for 5-50 degrees C, at pH 6.7. Under these conditions hisactophilin has moderate thermodynamic stability, and equilibrium and kinetic data are well fit by a two-state transition between the native and the denatured states. Equilibrium and kinetic m values decrease with increasing temperature, and decrease with increasing denaturant concentration. The betaF values at different temperatures and urea concentrations are quite constant, however, at about 0.7. This suggests that the transition state for hisactophilin unfolding is native-like and changes little with changing solution conditions, consistent with a narrow free energy profile for the transition state. The activation enthalpy and entropy of unfolding are unusually low for hisactophilin, as is also the case for the corresponding equilibrium parameters. Conventional Arrhenius and Eyring plots for both folding and unfolding are markedly non-linear, but these plots become linear for constant DeltaG/T contours. The Gibbs free energy changes for structural changes in hisactophilin have a non-linear denaturant dependence that is comparable to non-linearities observed for many other proteins. These non-linearities can be fit for many proteins using a variation of the Tanford model, incorporating empirical quadratic denaturant dependencies for Gibbs free energies of transfer of amino acid constituents from water to urea, and changes in fractional solvent accessible surface area of protein constituents based on the known protein structures. Noteworthy exceptions that are not well fit include amyloidogenic proteins and large proteins, which may form intermediates. The model is easily implemented and should be widely applicable to analysis of urea-induced structural transitions in proteins.     

Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae.

Protein unfolding of eight different phycocyanins was investigated utilizing circular dichroism and visible spectra. The phycocyanin samples were extracted from algae that are normally found in vastly different environments, and are classified as mesophilic, thermophilic, halophilic and psychrophilic. The ability of these proteins to resist the denaturant urea is in the order of thermophile greater than mesophile, halophile greater than psychrophile. Based on a two-state approximation the apparent free energies of protein unfolding at zero urea denaturant concentration, deltaGH2Oapp, were found to range from 2.4 to 8.8 kcal/mole for the eight phycocyanins at pH 6 and 25 degrees C. The proteins from the thermophile are generally more stable than those from the mesophile. An extra stability of the halophile is believed due to the specific interaction of the proteins and the ions in solution. A correction for deltaGH2Oapp due to minor amino acid differences reveals that the stability and the structural properties of these proteins are primarily affected by this minor difference in amino acid compositions.     

Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at -80 degrees C than slow freezing at -20 degrees C. Initial cell concentration was another important factor, preferable for 1-3 x 10(10)CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.     

Lens crystallins and their microbial homologs: structure, stability, and function.

abg-Crystallins are the major protein components in the vertebrate eye lens--a as a molecular chaperone and b and g as structural proteins. Surprisingly, the latter two share some structural characteristics with a number of microbial stress proteins. The common denominator is not only the Greek key topology of their polypeptide chains but also their high intrinsic stability, which, in certain microbial crystallin homologs, is further enhanced by high-affinity Ca2+-binding. Recent studies of natural and mutant vertebrate bg-crystallins as well as spherulin 3a from Physarum polycephalum and Protein S from Myxococcus xanthus allowed the correlation of structure and stability of crystallins to be elucidated in some detail. From the thermodynamic point of view, stability increments come from (1) local interactions involved in the close packing of the cooperative units, (2) the all-b secondary structure of the Greek-key motif, (3) intramolecular interactions between domains, (4) intermolecular domain interactions, including 3D domain swapping and (v) excluded volume effects due to "molecular crowding" at the high cellular protein concentrations. Apart from these contributions to the Gibbs free energy of stability, significant kinetic stabilization originates from the high activation energy barrier determining the rate of unfolding from the native to the unfolded state. From the functional point of view, the high stability is responsible for the long-term transparency of the eye lens, on the one hand, and the stress resistance of the microorganisms in their dormant state on the other. Local structural perturbations due to chemical modification, wrong protein interactions, or other irreversible processes may lead to protein aggregation. A leading cataract hypothesis is that only after a-crystallin, a member of the small heat-shock protein family, is titrated out does pathological opacity occur. Understanding the structural basis of protein stability in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.     

Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations

A Raman spectrometer and dynamic light scattering system were combined in a single platform (Raman–DLS) to provide concomitant higher order structural and hydrodynamic size data for therapeutic proteins at high concentration. As model therapeutic proteins, we studied human serum albumin (HSA) and intravenous immunoglobulin (IVIG). HSA concentration and temperature interval during heating did not affect the onset temperatures for conformation perturbation or aggregation. The impact of pH on thermal stability of HSA was tested at pHs 3, 5, and 8. Stability was the greatest at pH 8, but distinct unfolding and aggregation behaviors were observed at the different pHs. HSA structural transitions and aggregation kinetics were also studied in real time during isothermal incubations at pH 7. In a forced oxidation study, it was found that hydrogen peroxide (H₂O₂) treatment reduced the thermal stability of HSA. Finally, the structure and thermal stability of IVIG were studied, and a comprehensive characterization of heating-induced structural perturbations and aggregation was obtained. In conclusion, by providing comprehensive data on protein tertiary and secondary structures and hydrodynamic size during real-time heating or isothermal incubation experiments, the Raman–DLS system offers unique physical insights into the properties of high-concentration protein samples.     

Comparison of the folding mechanism of highly homologous proteins in the lipid‐binding protein family

The folding mechanism of two closely related proteins in the intracellular lipid‐binding protein family, human bile acid‐binding protein (hBABP), and rat bile acid‐binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two‐state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6‐fluorotryptophan‐labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Characterizing intermolecular interactions that initiate native-like protein aggregation

Folded proteins can access aggregation-prone states without the need for transitions that cross the energy barriers for unfolding. In this study we characterized the initial steps of aggregation from a native-like state of the acylphosphatase from Sulfolobus solfataricus (Sso AcP). Using computer simulations restrained by experimental hydrogen/deuterium (H/D) exchange data, we provide direct evidence that under aggregation-promoting conditions Sso AcP populates a conformational ensemble in which native-like structure is retained throughout the sequence in the absence of local unfolding (N¹), although the protein exhibits an increase in hydrodynamic radius and dynamics. This transition leads an edge strand to experience an increased affinity for a specific unfolded segment of the protein. Direct measurements by means of H/D exchange rates, isothermal titration calorimetry, and intermolecular relaxation enhancements show that after formation of N¹, an intermolecular interaction with an antiparallel arrangement is established between the edge strand and the unfolded segment of the protein. However, under conditions that favor the fully native state of Sso AcP, such an interaction is not established. Thus, these results reveal a novel (to our knowledge) self-assembly mechanism for a folded protein that is based on the increased flexibility of highly aggregation-prone segments in the absence of local unfolding.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.