Effect of thermal treatments on the structural change and the hemostatic property of hair extracted proteins.

Human hair is a readily available source for hair protein-based biomaterial and is increasingly explored as an alternative to existing hemostatic materials. The hair protein is a complex mixture of multiple proteins, which are preferably extracted at relatively high temperatures (50-90 °C) for increasing protein yields. However, the effect of processing temperature on the hemostatic property of the hair derived proteins are not yet well-understood. The objective of the current study was to characterize the influence of thermal treatments (37 °C, 50 °C, 75 °C, 80 °C, and 90 °C) on the (i) secondary structure of different fractions of hair proteins including keratin (40-65 kDa) and keratin-associated proteins (KAPs, 6-30 kDa), and (ii) their capability to precipitate the soluble fibrinogen in an in vitro fibrin clotting assay. Our results indicated that the thermal treatments induced changes to the helical contents of hair-derived protein extracts and also increased the precipitation amount and rate of soluble fibrinogen. While further studies are required to better understand the exact role of hair protein fractions on the coagulation process, the current research suggests that the hair proteins extracted under relatively high temperatures is a prerequisite approach for improving the hemostatic property of human hair-derived proteins.

Site of Tagging Influences the Ochratoxin Recognition by Peptide NFO4: A Molecular Dynamics Study.

Molecular recognition by synthetic peptides is growing in importance in the design of biosensing elements used in the detection and monitoring of a wide variety of hapten bioanlaytes. Conferring specificity via bioimmobilization and subsequent recovery and purification of such sensing elements are aided by the use of affinity tags. However, the tag and its site of placement can potentially compromise the hapten recognition capabilities of the peptide, necessitating a detailed experimental characterization and optimization of the tagged molecular recognition entity. The objective of this study was to assess the impact of site-specific tags on a native peptide's fold and hapten recognition capabilities using an advanced molecular dynamics (MD) simulation approach involving bias-exchange metadynamics and Markov State Models. The in-solution binding preferences of affinity tagged NFO4 (VYMNRKYYKCCK) to chlorinated (OTA) and non-chlorinated (OTB) analogues of ochratoxin were evaluated by appending hexa-histidine tags (6× His-tag) to the peptide's N-terminus (NterNFO4) or C-terminus (CterNFO4), respectively. The untagged NFO4 (NFO4), previously shown to bind with high affinity and selectivity to OTA, served as the control. Results indicate that the addition of site-specific 6× His-tags altered the peptide's native fold and the ochratoxin binding mechanism, with the influence of site-specific affinity tags being most evident on the peptide's interaction with OTA. The tags at the N-terminus of NFO4 preserved the native fold and actively contributed to the nonbonded interactions with OTA. In contrast, the tags at the C-terminus of NFO4 altered the native fold and were agnostic in its nonbonded interactions with OTA. The tags also increased the penalty associated with solvating the peptide-OTA complex. Interestingly, the tags did not significantly influence the nonbonded interactions or the penalty associated with solvating the peptide-OTB complex. Overall, the combined contributions of nonbonded interaction and solvation penalty were responsible for the retention of the native hapten recognition capabilities in NterNFO4 and compromised native recognition capabilities in CterNFO4. Advanced MD approaches can thus provide structural and energetic insights critical to evaluate the impact of site-specific tags and may aid in the selection and optimization of the binding preferences of a specific biosensing element.

Evaluation of Ochratoxin Recognition by Peptides Using Explicit Solvent Molecular Dynamics.

Biosensing platforms based on peptide recognition provide a cost-effective and stable alternative to antibody-based capture and discrimination of ochratoxin-A (OTA) vs. ochratoxin-B (OTB) in monitoring bioassays. Attempts to engineer peptides with improved recognition efficacy require thorough structural and thermodynamic characterization of the binding-competent conformations. Classical molecular dynamics (MD) approaches alone do not provide a thorough assessment of a peptide's recognition efficacy. In this study, in-solution binding properties of four different peptides, a hexamer (SNLHPK), an octamer (CSIVEDGK), NFO4 (VYMNRKYYKCCK), and a 13-mer (GPAGIDGPAGIRC), which were previously generated for OTA-specific recognition, were evaluated using an advanced MD simulation approach involving accelerated configurational search and predictive modeling. Peptide configurations relevant to ochratoxin binding were initially generated using biased exchange metadynamics and the dynamic properties associated with the in-solution peptide-ochratoxin binding were derived from Markov State Models. Among the various peptides, NFO4 shows superior in-solution OTA sensing and also shows superior selectivity for OTA vs. OTB due to the lower penalty associated with solvating its bound complex. Advanced MD approaches provide structural and energetic insights critical to the hapten-specific recognition to aid the engineering of peptides with better sensing efficacies.

Cluster analysis of molecular simulation trajectories for systems where both conformation and orientation of the sampled states are important.

Clustering methods have been widely used to group together similar conformational states from molecular simulations of biomolecules in solution. For applications such as the interaction of a protein with a surface, the orientation of the protein relative to the surface is also an important clustering parameter because of its potential effect on adsorbed-state bioactivity. This study presents cluster analysis methods that are specifically designed for systems where both molecular orientation and conformation are important, and the methods are demonstrated using test cases of adsorbed proteins for validation. Additionally, because cluster analysis can be a very subjective process, an objective procedure for identifying both the optimal number of clusters and the best clustering algorithm to be applied to analyze a given dataset is presented. The method is demonstrated for several agglomerative hierarchical clustering algorithms used in conjunction with three cluster validation techniques. © 2016 Wiley Periodicals, Inc.

Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-ß-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination.

Parameterization of an interfacial force field for accurate representation of peptide adsorption free energy on high-density polyethylene.

Interfacial force field (IFF) parameters for use with the CHARMM force field have been developed for interactions between peptides and high-density polyethylene (HDPE). Parameterization of the IFF was performed to achieve agreement between experimental and calculated adsorption free energies of small TGTG-X-GTGT host-guest peptides (T = threonine, G = glycine, and X = variable amino-acid residue) on HDPE, with ±0.5 kcal/mol agreement. This IFF parameter set consists of tuned nonbonded parameters (i.e., partial charges and Lennard-Jones parameters) for use with an in-house-modified CHARMM molecular dynamic program that enables the use of an independent set of force field parameters to control molecular behavior at a solid-liquid interface. The R correlation coefficient between the simulated and experimental peptide adsorption free energies increased from 0.00 for the standard CHARMM force field parameters to 0.88 for the tuned IFF parameters. Subsequent studies are planned to apply the tuned IFF parameter set for the simulation of protein adsorption behavior on an HDPE surface for comparison with experimental values of adsorbed protein orientation and conformation.

Experimental characterization of adsorbed protein orientation, conformation, and bioactivity.

Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems.

Adsorption-induced changes in ribonuclease A structure and enzymatic activity on solid surfaces.

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy.

Protein helical structure determination using CD spectroscopy for solutions with strong background absorbance from 190 to 230nm.

Conventional empirical methods for the quantification of the helical content of proteins in solution using circular dichroism (CD) primarily rely on spectral data acquired between wavelengths of 190 and 230nm. The presence of chemical species in a protein solution with strong absorbance within this range can interfere with the ability to use these methods for the determination of the protein's helical structure. The objective of this research was to overcome this problem by developing a method for CD spectral analysis that relies on spectral features above this wavelength range. In this study, we determined that the slopes of CD spectra acquired over the 230 to 240nm region strongly correlate with the helix contents including α-helix and 310-helix of protein as determined using conventional CD algorithms that rely on wavelengths between 190 and 230nm. This approach (i.e., the 230-240nm slope method) is proposed as an effective method to determine the helix content within proteins in the presence of additives such as detergents or denaturants with high absorbance of wavelengths up to 230nm.

Determination of orientation and adsorption-induced changes in the tertiary structure of proteins on material surfaces by chemical modification and peptide mapping.

The labeling of amino acid residues followed by peptide mapping via mass spectrometry (AAL/MS) is a promising technique to provide detailed information on the adsorption-induced changes in its solvent accessibility. However, the potential of this method for the study of adsorbed protein structure is largely undeveloped at this time. The objective of this research was therefore to extend these capabilities by developing and applying AAL/MS techniques for a range of amino acid types to identify the dominant configurations of an adsorbed protein on a material surface. In this study, the configuration of hen egg white lysozyme (HEWL) adsorbed on fused silica glass, high-density polyethylene (HDPE) and poly(methyl methacrylate) (PMMA) was mapped by combining the labeling profiles obtained from five amino acid labels, which were independently applied. In order to be able to combine the results from the different amino acid labeling processes, the intensity of the HEWL segment without the target amino acids was used as an internal control to normalize the intensity shifts to an equivalent level. The resulting quantitative differences in the normalized amino acid profiles were then used to provide insights into adsorbed orientation, protein-protein interactions and adsorption-induced tertiary unfolding of HEWL, which were found to be distinctly different between the fused silica glass, HDPE and PMMA surfaces. The developed technique has the potential for broad application and for expansion to additional targeted amino acids to provide highly detailed information on the adsorbed state of any protein on any given surface.

Quantification of the influence of protein-protein interactions on adsorbed protein structure and bioactivity.

While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2h to saturate the surface, followed by immersion in pure buffer solution for 15h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein's secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL's structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL's bioactivity on this surface, such as the accessibility of HEWL's bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein.

Peptide-surface adsorption free energy comparing solution conditions ranging from low to medium salt concentrations.

Multi-technique methods involving surface plasmon resonance spectroscopy and atomic force microscopy provide experimental data for the characterization of peptide adsorption on self-assembled monolayers. A comparative study is carried out in phosphate-buffered saline (PBS) and potassium phosphate-buffered (PPB) water to determine the influence of the salt concentration on the adsorption behavior (see figure; ΔG(0)(ads) : free energy of peptide adsorption, F(des) : force required for peptide desorption).

Development of a tuned interfacial force field parameter set for the simulation of protein adsorption to silica glass.

Adsorption free energies for eight host-guest peptides (TGTG-X-GTGT, with X = N, D, G, K, F, T, W, and V) on two different silica surfaces [quartz (100) and silica glass] were calculated using umbrella sampling and replica exchange molecular dynamics and compared with experimental values determined by atomic force microscopy. Using the CHARMM force field, adsorption free energies were found to be overestimated (i.e., too strongly adsorbing) by about 5-9 kcal/mol compared to the experimental data for both types of silica surfaces. Peptide adsorption behavior for the silica glass surface was then adjusted using a modified version of the CHARMM program, which we call dual force-field CHARMM, which allows separate sets of nonbonded parameters (i.e., partial charge and Lennard-Jones parameters) to be used to represent intra-phase and inter-phase interactions within a given molecular system. Using this program, interfacial force field (IFF) parameters for the peptide-silica glass systems were corrected to obtain adsorption free energies within about 0.5 kcal/mol of their respective experimental values, while IFF tuning for the quartz (100) surface remains for future work. The tuned IFF parameter set for silica glass will subsequently be used for simulations of protein adsorption behavior on silica glass with greater confidence in the balance between relative adsorption affinities of amino acid residues and the aqueous solution for the silica glass surface.

Determination of peptide-surface adsorption free energy for material surfaces not conducive to SPR or QCM using AFM.

The interactions between peptides and proteins with material surfaces are of primary importance in many areas of biotechnology. While surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance (QCM) methods have proven to be very useful in measuring fundamental properties characterizing adsorption behavior, such as the free energy of adsorption for peptide-surface interactions, these methods are largely restricted to use for materials that can readily form nanoscale-thick films over the respective sensor surfaces. Many materials including most polymers, ceramics, and inorganic glasses, however, are not readily suitable for use with SPR or QCM methods. To overcome these limitations, we recently showed that desorption forces (F(des)) obtained using a standardized AFM method linearly correlate to standard-state adsorption free energy values (ΔG°(ads)) measured from SPR in phosphate buffered saline (PBS: phosphate buffered 140 mM NaCl, pH 7.4). This approach thus provides a means to determine ΔG°(ads) for peptide adsorption using AFM that can be applied to any flat material surface. In this present study, we investigated the F(des)-ΔG°(ads) correlation between AFM and SPR data in PBS for a much broader range of systems including eight different types of peptides on a set of eight different alkanethiol self-assembled monolayer (SAM) surfaces. The resulting correlation was then used to estimate ΔG°(ads) from F(des) determined by AFM for selected bulk polymer and glass/ceramic materials such as poly(methyl methacrylate) (PMMA), high-density polyethylene (HDPE), fused silica glass, and a quartz (100) surface. The results of these studies support our previous findings regarding the strong correlation between F(des) measured by AFM and ΔG°(ads) determined by SPR, and provides a means to estimate ΔG°(ads) for peptide adsorption on macroscopically thick samples of materials that are not conducive for use with SPR or QCM.