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.

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.

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.

TIGER2 with solvent energy averaging (TIGER2A): An accelerated sampling method for large molecular systems with explicit representation of solvent.

The recently developed "temperature intervals with global exchange of replicas" (TIGER2) accelerated sampling method is found to have inaccuracies when applied to systems with explicit solvation. This inaccuracy is due to the energy fluctuations of the solvent, which cause the sampling method to be less sensitive to the energy fluctuations of the solute. In the present work, the problem of the TIGER2 method is addressed in detail and a modification to the sampling method is introduced to correct this problem. The modified method is called "TIGER2 with solvent energy averaging," or TIGER2A. This new method overcomes the sampling problem with the TIGER2 algorithm and is able to closely approximate Boltzmann-weighted sampling of molecular systems with explicit solvation. The difference in performance between the TIGER2 and TIGER2A methods is demonstrated by comparing them against analytical results for simple one-dimensional models, against replica exchange molecular dynamics (REMD) simulations for sampling the conformation of alanine dipeptide and the folding behavior of (AAQAA)3 peptide in aqueous solution, and by comparing their performance in sampling the behavior of hen egg-white lysozyme in aqueous solution. The new TIGER2A method solves the problem caused by solvent energy fluctuations in TIGER2 while maintaining the two important characteristics of TIGER2, i.e., (1) using multiple replicas sampled at different temperature levels to help systems efficiently escape from local potential energy minima and (2) enabling the number of replicas used for a simulation to be independent of the size of the molecular system, thus providing an accelerated sampling method that can be used to efficiently sample systems considered too large for the application of conventional temperature REMD.

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.

Simulation of multiphase systems utilizing independent force fields to control intraphase and interphase behavior.

Fixed-charge empirical force fields have been developed and widely used over the past three decades for all-atom molecular simulations. Most simulation programs providing these methods enable only one set of force field parameters to be used for the entire system. Whereas this is generally suitable for single-phase systems, the molecular environment at the interface between two phases may be sufficiently different from the individual phases to require a different set of parameters to be used to accurately represent the system. Recently published simulations of peptide adsorption to material surfaces using the CHARMM force field have clearly demonstrated this issue by revealing that calculated values of adsorption free energy substantially differ from experimental results. Whereas nonbonded parameters could be adjusted to correct this problem, this cannot be done without also altering the conformational behavior of the peptide in solution, for which CHARMM has been carefully tuned. We have developed a dual-force-field approach (Dual-FF) to address this problem and implemented it in the CHARMM simulation package. This Dual-FF method provides the capability to use two separate sets of nonbonded force field parameters within the same simulation: one set to represent intraphase interactions and a separate set to represent interphase interactions. Using this approach, we show that interfacial parameters can be adjusted to correct errors in peptide adsorption free energy without altering peptide conformational behavior in solution. This program thus provides the capability to enable both intraphase and interphase molecular behavior to be accurately and efficiently modeled in the same simulation.

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).

Time-dependent conformational changes in adsorbed albumin and its effect on platelet adhesion.

Recent studies have shown that platelets can adhere to adsorbed albumin (Alb) through a receptor-mediated mechanism, but only if the Alb undergoes more than a critical degree of adsorption-induced unfolding. The objectives of this research were to investigate whether Alb that was initially adsorbed in a manner that induced unfolding that was less than this critical level would undergo further unfolding with time and, if so, whether this would induce the onset of platelet adhesion once this critical level was exceeded. To address these questions, CD spectropolarimetry was used to monitor the structure of Alb on OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces, with the Alb initially adsorbed under conditions resulting in degrees of unfolding that were below this critical level, and then the adsorbed Alb layers were aged over 6 months in sterile physiological saline at 37 °C. Platelet adhesion to Alb was quantified at selected time points via a lactate dehydrogenase (LDH) assay. The results indicate that an adsorbed Alb layer does undergo further structural changes with increasing residence time and supports platelet adhesion once it unfolds beyond the previously determined critical level. These results may be relevant to the clinically observed problem of the onset of late-thrombosis, which occurs on cardiovascular implants such as drug-eluting stents.

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.

The temperature intervals with global exchange of replicas empirical accelerated sampling method: parameter sensitivity and extension to a complex molecular system.

The recently developed "temperature intervals with global exchange of replicas" (TIGER2) algorithm is an efficient replica-exchange sampling algorithm that provides the freedom to specify the number of replicas and temperature levels independently of the size of the system and temperature range to be spanned, thus making it particularly well suited for sampling molecular systems that are considered to be too large to be sampled using conventional replica exchange methods. Although the TIGER2 method is empirical in nature, when appropriately applied it is able to provide sampling that satisfies the balance condition and closely approximates a Boltzmann-weighted ensemble of states. In this work, we evaluated the influence of factors such as temperature range, temperature spacing, replica number, and sampling cycle design on the accuracy of a TIGER2 simulation based on molecular dynamics simulations of alanine dipeptide in implicit solvent. The influence of these factors is further examined by calculating the properties of a complex system composed of the B1 immunoglobulin-binding domain of streptococcal protein G (protein G) in aqueous solution. The accuracy of a TIGER2 simulation is particularly sensitive to the maximum temperature level selected for the simulation. A method to determine the appropriate maximum temperature level to be used in a TIGER2 simulation is presented.

Correlation between desorption force measured by atomic force microscopy and adsorption free energy measured by surface plasmon resonance spectroscopy for peptide-surface interactions.

Surface plasmon resonance (SPR) spectroscopy is a useful technique for thermodynamically characterizing peptide-surface interactions; however, its usefulness is limited to the types of surfaces that can readily be formed as thin layers on the nanometer scale on metallic biosensor substrates. Atomic force microscopy (AFM), on the other hand, can be used with any microscopically flat surface, thus making it more versatile for studying peptide-surface interactions. AFM, however, has the drawback of data interpretation due to questions regarding peptide-to-probe-tip density. This problem could be overcome if results from a standardized AFM method could be correlated with SPR results for a similar set of peptide-surface interactions so that AFM studies using the standardized method could be extended to characterize peptide-surface interactions for surfaces that are not amenable for characterization by SPR. In this article, we present the development and application of an AFM method to measure adsorption forces for host-guest peptides sequence on surfaces consisting of alkanethiol self-assembled monolayers (SAMs) with different functionality. The results from these studies show that a linear correlation exists between these data and the adsorption free energy (ΔG(o)(ads)) values associated with a similar set of peptide-surface systems available from SPR measurements. These methods will be extremely useful to characterize thermodynamically the adsorption behavior for peptides on a much broader range of surfaces than can be used with SPR to provide information related to understanding protein adsorption behavior to these surfaces and to provide an experimental database that can be used for the evaluation, modification, and validation of force field parameters that are needed to represent protein adsorption behavior accurately for molecular simulations.

XPS and ToF-SIMS investigation of alpha-helical and beta-strand peptide adsorption onto SAMs.

14-mer alpha-helix and 15-mer beta-strand oligopeptides composed of leucine (L) and lysine (K) were used to investigate peptide adsorption and orientation onto well-defined methyl and carboxylic acid-terminated self-assembled monolayer (SAM) surfaces with X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). XPS showed that both peptides reached monolayer thickness on both SAMs, but significantly higher solution concentrations were required to reach this coverage on the methyl SAMs. This shows that the peptides adsorb more strongly onto the carboxyl-terminated SAMs. The excess oxygen detected by XPS and the H(3)O(+) signal detected by ToF-SIMS for the SAMs with adsorbed peptides indicated that water molecules are associated with the adsorbed peptides, even under ultrahigh-vacuum conditions. Changes in the number of L and K fragments detected by ToF-SIMS indicate that the beta-strand oriented differently on the two SAMs. The L side chains were preferentially associated with the methyl-terminated SAM, and the K side chains were preferentially associated with the carboxyl SAM. In contrast, little change in the ToF-SIMS K/L ratio was observed for the alpha-helix peptide absorbed on the two SAMs, indicating that ToF-SIMS was not as sensitive to the orientation of the alpha-helix peptide.

Assessment of the transferability of a protein force field for the simulation of peptide-surface interactions.

In order to evaluate the transferability of existing empirical force fields for all-atom molecular simulations of protein adsorption behavior, we have developed and applied a method to calculate the adsorption free energy (DeltaG(ads)) of model peptides on functionalized surfaces for comparison with available experimental data. Simulations were conducted using the CHARMM program and force field using a host-guest peptide with the sequence TGTG-X-GTGT (where G and T are glycine and threonine amino acid residues, respectively, with X representing valine, threonine, aspartic acid, phenylalanine or lysine) over nine different functionalized alkanethiol self-assembled monolayer (SAM) surfaces with explicitly represented solvent. DeltaG(ads) was calculated using biased-energy replica exchange molecular dynamics to adequately sample the conformational states of the system. The simulation results showed that the CHARMM force-field was able to represent DeltaG(ads) within 1 kcal/mol of the experimental values for most systems, while deviations as large as 4 kcal/mol were found for others. In particular, the simulations reveal that CHARMM underestimates the strength of adsorption on the hydrophobic and positively charged amine surfaces. These results clearly show that improvements in force field parameterization are needed in order to accurately represent interactions between amino acid residues and functional groups of a surface and they provide a means for force field evaluation and modification for the eventual development and validation of an interfacial force field for the accurate simulation of protein adsorption behavior.

Fundamental Principles of the Thermodynamics and Kinetics of Protein Adsorption to Material Surfaces.

Protein adsorption is important for essentially any process that involves the contact of a protein-containing solution and a material surface, with the resulting formation of the adsorbed layer of protein determined by the thermodynamics and kinetics of the system involved. This paper presents an overview of the fundamentals of these processes. First, the hierarchical structure of proteins and the types of bonding that stabilize a protein's native-state structure are presented. This section is then followed by a section presenting the thermodynamic driving forces that influence the way that proteins adsorb and conformationally change for three characteristically different types of surface chemistries: nonpolar (hydrophobic) surfaces, neutral hydrophilic surfaces, and charged surfaces. The final section of this paper addresses how kinetics and thermodynamics combine together to influence protein adsorption behavior, followed by concluding remarks.

Assessing the influence of adsorbed-state conformation on the bioactivity of adsorbed enzyme layers.

Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for the fabrication of stable, reusable substrates with highly specific functionality. The performance of these systems is greatly dependent upon the orientation and conformation of the adsorbed enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces using circular dichroism (CD) spectroscopy and evaluate their bioactivity using colorimetric assays. These combined measurements provide molecular-level insights regarding whether observed changes in adsorbed enzyme bioactivity are due to the adsorbed orientation of an enzyme or adsorption-induced changes in its conformation. Using this approach, we investigated the adsorption behavior of lysozyme (HEWL), xylanase (XYL), and glucose oxidase (GOx) on OH-, CH(3)-, NH(2)-, and COOH-terminated alkanethiol self-assembled monolayer (SAM) surfaces. The bioactivities of small enzymes HEWL and XYL had pronounced variations between the different SAM surfaces despite their structural stability, highlighting the role of adsorbed orientation on bioactivity. In contrast, GOx, which is a much larger enzyme, exhibited wide variations in both its structure and bioactivity after adsorption, with adsorption-induced conformational changes actually enhancing its bioactivity. These results provide new insights into protein-surface interactions at the molecular level and demonstrate that adsorption can either promote or inhibit bioactivity depending on how the surface chemistry influences the orientation and conformational state of the enzyme on the surface.

Saccharide polymer brushes to control protein and cell adhesion to titanium.

Attaining control over the surface chemistry of titanium is critical to its use in medical implants, especially to address complications such as infection and loosening of implants over time, which still present significant challenges. The surface-initiated atom transfer radical polymerization (SI-ATRP) of a saccharide-substituted methacrylate, 2-gluconamidoethyl methacrylate (GAMA), affords dense polymer brushes that resist protein adsorption and cell adhesion. We further tailored the nature of the surfaces by covalent attachment of an adhesion peptide to afford control over cell adhesion. Whereas unmodified poly(GAMA) brushes prevent cell adhesion, brushes with a tethered GFOGER-containing peptide sequence promote the deposition of confluent well-spread cells. The presentation of adhesion proteins on a robust bioresistive background in this fashion constitutes a versatile approach to the development of new biomaterials.

TIGER2: an improved algorithm for temperature intervals with global exchange of replicas.

An empirical sampling method for molecular simulation based on "temperature intervals with global exchange of replicas" (TIGER2) has been developed to reduce the high demand for computational resources and the low computational efficiency of the conventional replica-exchange molecular dynamics (REMD) method. This new method overcomes the limitation of its previous version, called TIGER, which requires the assumption of constant heat capacity during quenching of replicas from elevated temperatures to the baseline temperature. The robustness of the TIGER2 method is examined by comparing it against a Metropolis Monte Carlo simulation for sampling the conformational distribution of a single butane molecule in vacuum, a REMD simulation for sampling the behavior of alanine dipeptide in explicit solvent, and REMD simulations for sampling the folding behavior of two peptides, (AAQAA)(3) and chignolin, in implicit solvent. The agreement between the results from these conventional sampling methods and the TIGER2 simulations indicates that the TIGER2 algorithm is able to closely approximate a Boltzmann-weighted ensemble of states for these systems but without the limiting assumptions that were required for the original TIGER algorithm. TIGER2 is an efficient replica-exchange sampling method that enables the number of replicas that are used for a replica-exchange simulation to be substantially reduced compared to the conventional REMD method.

A systematic procedure to build a relaxed dense-phase atomistic representation of a complex amorphous polymer using a coarse-grained modeling approach.

A systematic procedure has been developed to construct a relaxed dense-phase atomistic structure of a complex amorphous polymer. The numerical procedure consists of (1) coarse graining the atomistic model of the polymer into a mesoscopic model based on an iterative algorithm for potential inversion from distribution functions of the atomistic model, (2) relaxation of the coarse grained chain using a molecular dynamics scheme, and (3) recovery of the atomistic structure by reverse mapping based on the superposition of atomistic counterparts on the corresponding coarse grained coordinates. These methods are demonstrated by their application to construct a relaxed, dense-phase model of poly(DTB succinate), which is an amorphous tyrosine-derived biodegradable polymer that is being developed for biomedical applications. Both static and dynamic properties from the coarse-grained and atomistic simulations are analyzed and compared. The coarse-grained model, which contains the essential features of the DTB succinate structure, successfully described both local and global structural properties of the atomistic chain. The effective speedup compared to the corresponding atomistic simulation is substantially above 10(2), thus enabling simulation times to reach well into the characteristic experimental regime. The computational approach for reversibly bridging between coarse-grained and atomistic models provides an efficient method to produce relaxed dense-phase all-atom molecular models of complex amorphous polymers that can subsequently be used to study and predict the atomistic-level behavior of the polymer under different environmental conditions in order to optimally design polymers for targeted applications.

In Vitro Thrombogenicity Testing of Biomaterials.

The short- and long-term thrombogenicity of implant materials is still unpredictable, which is a significant challenge for the treatment of cardiovascular diseases. A knowledge-based approach for implementing biofunctions in materials requires a detailed understanding of the medical device in the biological system. In particular, the interplay between material and blood components/cells as well as standardized and commonly acknowledged in vitro test methods allowing a reproducible categorization of the material thrombogenicity requires further attention. Here, the status of in vitro thrombogenicity testing methods for biomaterials is reviewed, particularly taking in view the preparation of test materials and references, the selection and characterization of donors and blood samples, the prerequisites for reproducible approaches and applied test systems. Recent joint approaches in finding common standards for a reproducible testing are summarized and perspectives for a more disease oriented in vitro thrombogenicity testing are discussed.

The blood compatibility challenge. Part 2: Protein adsorption phenomena governing blood reactivity.

The adsorption of proteins is the initiating event in the processes occurring when blood contacts a "foreign" surface in a medical device, leading inevitably to thrombus formation. Knowledge of protein adsorption in this context has accumulated over many years but remains fragmentary and incomplete. Moreover, the significance and relevance of the information for blood compatibility are not entirely agreed upon in the biomaterials research community. In this review, protein adsorption from blood is discussed under the headings "agreed upon" and "not agreed upon or not known" with respect to: protein layer composition, effects on coagulation and complement activation, effects on platelet adhesion and activation, protein conformational change and denaturation, prevention of nonspecific protein adsorption, and controlling/tailoring the protein layer composition. STATEMENT OF SIGNIFICANCE: This paper is part 2 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.