Exploring the energy landscape of a molecular engineered analog of a tumor-homing peptide

Recently a new non-coded amino acid was designed as a replacement for Arg, to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This constrained Arg analog, denoted c(5)Arg, was engineered to also promote the stability of the CREKA bioactive conformation. The conformational profile of the CREKA analog obtained by replacing Arg by c(5)Arg has been extensively investigated in this work. Two molecular dynamics simulations-based strategies have been employed: a modified simulated annealing and replica exchange. Results obtained using both techniques show that the conformational features of the new analog fulfill the purpose of its design. The new CREKA analog not only preserves the main structural attributes found for the bioactive conformation of the parent peptide but also shows lower flexibility. Moreover, the conformational profile of the mutated peptide narrows towards the most stable structures previously observed for the parent CREKA peptide.     

Engineering strategy to improve peptide analogs: from structure-based computational design to tumor homing

We present a chemical strategy to engineer analogs of the tumor-homing peptide CREKA (Cys-Arg-Glu-Lys-Ala), which binds to fibrin and fibrin-associated clotted plasma proteins in tumor vessels (Simberg et al. in Proc Natl Acad Sci USA 104:932–936, 2007) with improved ability to inhibit tumor growth. Computer modeling using a combination of simulated annealing and molecular dynamics were carried out to design targeted replacements aimed at enhancing the stability of the bioactive conformation of CREKA. Because this conformation presents a pocket-like shape with the charged groups of Arg, Glu and Lys pointing outward, non-proteinogenic amino acids α-methyl and N-methyl derivatives of Arg, Glu and Lys were selected, rationally designed and incorporated into CREKA analogs. The stabilization of the bioactive conformation predicted by the modeling for the different CREKA analogs matched the tumor fluorescence results, with tumor accumulation increasing with stabilization. Here we report the modeling, synthetic procedures, and new biological assays used to test the efficacy and utility of the analogs. Combined, our results show how studies based on multi-disciplinary collaboration can converge and lead to useful biomedical advances.     

A simulation strategy for the atomistic modeling of flexible molecules covalently tethered to rigid surfaces: Application to peptides

A computational strategy to model flexible molecules tethered to a rigid inert surface is presented. The strategy is able to provide uncorrelated relaxed microstructures at the atomistic level. It combines an algorithm to generate molecules tethered to the surface without atomic overlaps, a method to insert solvent molecules and ions in the simulation box, and a powerful relaxation procedure. The reliability of the strategy has been investigated by simulating two different systems: (i) mixed monolayers consisting of binary mixtures of long‐chain alkyl thiols of different lengths adsorbed on a rigid inert surface and (ii) CREKA (Cys‐Arg‐Glu‐Lys‐Ala), a short linear pentapeptide that recognizes clotted plasma proteins and selectively homes to tumors, covalently tethered to a rigid inert surface in aqueous solution. In the first, we examined the segregation of the two species in the monolayers using different long‐chain:short‐chain ratios, whereas in the second, we explored the conformational space of CREKA and ions distribution considering densities of peptides per nm² ranging from 0.03 to 1.67. Results indicate a spontaneous segregation in alkyl thiol monolayers, which enhances when the concentration of longest chains increases. However, the whole conformational profile of CREKA depends on the number of molecules tethered to the surface pointing out the large influence of molecular density on the intermolecular interactions, even though the bioactive conformation was found as the most stable in all cases. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Effect of poly(ethylene glycol) (PEG) spacers on the conformational properties of small peptides: a molecular dynamics study

Poly(ethylene glycol) (PEG) is used as an inert spacer in a wide range of biotechnological applications such as to display peptides and proteins on surfaces for diagnostic purposes. In such applications it is critical that the peptide is accessible to solvent and that the PEG does not affect the conformational properties of the peptide to which it is attached. Using molecular dynamics (MD) simulation techniques, we have investigated the influence of a commonly used PEG spacer on the conformation properties of a series of five peptides with differing physical-chemical properties (YGSLPQ, VFVVFV, GSGGSG, EEGEEG, and KKGKKG). The conformational properties of the peptides were compared (a) free in solution, (b) attached to a PEG-11 spacer in solution, and (c) constrained to a two-dimensional lattice via a (PEG-11)(3) spacer, mimicking a peptide displayed on a surface as used in microarray techniques. The simulations suggest that the PEG spacer has little effect on the conformational properties of small neutral peptides but has a significant effect on the conformational properties of small highly charged peptides. When constrained to a two-dimensional surface at peptide densities similar to those used experimentally, it was found that the peptides, in particular the polar and nonpolar peptides, aggregated strongly. The peptides also partitioned into the PEG layer. Potentially, this means that at high packing densities only a small fraction of the peptide attached to the surface would in fact be accessible to a potential interaction partner.     

Fibrin Association at Hybrid Biointerfaces Made of Clot‐Binding Peptides and Polythiophene

The properties as biointerfaces of electroactive conducting polymer–peptide biocomposites formed by poly(3,4‐ethylenedioxythiophene) (PEDOT) and CREKA or CR(NMe)EKA peptide sequences (where Glu has been replaced by N‐methyl‐Glu in the latter) have been compared. CREKA is a linear pentapeptide that recognizes clotted plasma proteins and selectively homes to tumors, while CR(NMe)EKA is an engineer to improve such properties by altering peptide–fibrin interactions. Differences between PEDOT‐CREKA and PEDOT‐CR(NMe)EKA reflect dissemblance in the organization of the peptides into the polymeric matrix. Both peptides affect fibrinogen thrombin‐catalyzed polymerization causing the immediate formation of fibrin, whereas in the absence of thrombin this phenomenon is only observed for CR(NMe)EKA. Consistently, the fibrin‐adsorption capacity is higher for PEDOT‐CR(NMe)EKA than for PEDOT‐CREKA, even though in both cases adsorbed fibrin exhibits round‐like morphologies rather than the characteristic fibrous structure. PEDOT‐peptide films coated with fibrin are selective in terms of cell adhesion, promoting the attachment of metastatic cells with respect to normal cells.

     

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. © 2012 Wiley Periodicals, Inc.     

Lattice models of peptide aggregation: evaluation of conformational search algorithms

We present a series of conformational search calculations on the aggregation of short peptide fragments that form fibrils similar to those seen in many protein mis-folding diseases. The proteins were represented by a face-centered cubic lattice model with the conformational energies calculated using the Miyazawa-Jernigan potential. The searches were performed using algorithms based on the Metropolis Monte Carlo method, including simulated annealing and replica exchange. We also present the results of searches using the tabu search method, an algorithm that has been used for many optimization problems, but has rarely been used in protein conformational searches. The replica exchange algorithm consistently found more stable structures then the other algorithms, and was particularly effective for the octamers and larger systems.     

Convergence and sampling efficiency in replica exchange simulations of peptide folding in explicit solvent

Replica exchange methods (REMs) are increasingly used to improve sampling in molecular dynamics (MD) simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD (T-REMD) simulations of a beta-heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated (275 K). However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature (300 K) the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on (random) fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems.     

Efficient evaluation of sampling quality of molecular dynamics simulations by clustering of dihedral torsion angles and Sammon mapping

A direct conformational clustering and mapping approach for peptide conformations based on backbone dihedral angles has been developed and applied to compare conformational sampling of Met-enkephalin using two molecular dynamics (MD) methods. Efficient clustering in dihedrals has been achieved by evaluating all combinations resulting from independent clustering of each dihedral angle distribution, thus resolving all conformational substates. In contrast, Cartesian clustering was unable to accurately distinguish between all substates. Projection of clusters on dihedral principal component (PCA) subspaces did not result in efficient separation of highly populated clusters. However, representation in a nonlinear metric by Sammon mapping was able to separate well the 48 highest populated clusters in just two dimensions. In addition, this approach also allowed us to visualize the transition frequencies between clusters efficiently. Significantly, higher transition frequencies between more distinct conformational substates were found for a recently developed biasing-potential replica exchange MD simulation method allowing faster sampling of possible substates compared to conventional MD simulations. Although the number of theoretically possible clusters grows exponentially with peptide length, in practice, the number of clusters is only limited by the sampling size (typically much smaller), and therefore the method is well suited also for large systems. The approach could be useful to rapidly and accurately evaluate conformational sampling during MD simulations, to compare different sampling strategies and eventually to detect kinetic bottlenecks in folding pathways.     

Shedding light on biomolecule conformational dynamics using fluorescence measurements of trapped ions

Biomolecule conformational change has been widely investigated in solution using several methods; however, much less experimental data about structural changes are available for completely isolated, gas-phase biomolecules. Studies of conformational change in unsolvated biomolecules are required to complement the interpretation of mass spectrometry measurements and in addition, can provide a means to directly test theoretical simulations of biomolecule structure and dynamics independent of a simulated solvent. In this Feature Article, we review our recent introduction of a fluorescence-based method for probing local conformational dynamics in unsolvated biomolecules through interactions of an attached dye with tryptophan (Trp) residues and fields originating on charge sites. Dye-derivatized biomolecule ions are formed by electrospray ionization and are trapped in a variable-temperature quadrupole ion trap in which they are irradiated with either continuous or short pulse lasers to excite fluorescence. Fluorescence is measured as a function of temperature for different charge states. Optical measurements of the dye fluorescence include average intensity changes, changes in the emission spectrum, and time-resolved measurements of the fluorescence decay. These measurements have been applied to the miniprotein, Trp-cage, polyproline peptides and to a beta-hairpin-forming peptide, and the results are presented as examples of the broad applicability and utility of these methods. Model fits to Trp-cage fluorescence data measured as a function of temperature provide quantitative information on the thermodynamics of conformational changes, which are reproduced well by molecular dynamics. Time-resolved measurements of the fluorescence decays of Trp-cage and small polyproline peptides definitively demonstrate the occurrence of fluorescence quenching by the amino acid Trp in unsolvated biomolecules.     

Conformational memories with variable bond angles

Conformational Memories (CM) is a simulated annealing/Monte Carlo method that explores peptide and protein dihedral conformational space completely and efficiently, independent of the original conformation. Here we extend the CM method to include the variation of a randomly chosen bond angle, in addition to the standard variation of two or three randomly chosen dihedral angles, in each Monte Carlo trial of the CM exploratory and biased phases. We test the hypothesis that the inclusion of variable bond angles in CM leads to an improved sampling of conformational space. We compare the results with variable bond angles to CM with no bond angle variation for the following systems: (1) the pentapeptide Met-enkephalin, which is a standard test case for conformational search methods; (2) the proline ring pucker in a 17mer model peptide, (Ala)(8)Pro(Ala)(8); and (3) the conformations of the Ser 7.39 chi(1) in transmembrane helix 7 (TMH7) of the cannabinoid CB1 receptor, a 25-residue system. In each case, analysis of the CM results shows that the inclusion of variable bond angles results in sampling of regions of conformational space that are inaccessible to CM calculations with only variable dihedral angles, and/or a shift in conformational populations from those calculated when variable bond angles are not included. The incorporation of variable bond angles leads to an improved sampling of conformational space without loss of efficiency. Our examples show that this improved sampling leads to better exploration of biologically relevant conformations that have been experimentally validated.     

Conformational analysis of a stereochemically complete set of cis-enediol peptide analogues

A conformational analysis of a stereochemically complete set of peptide analogues based on a cis-enediol unit is presented. The cis-enediol unit, which can replace a two or a three amino acid segment of a peptide, contains two "side chains", four asymmetrical carbon atoms, and six free dihedral angles. To determine the accessible conformational space, the molecules are divided into three fragments, each containing two free dihedral angles. The energy surfaces are computed for all dihedral angle values, and the possible conformations of the cis-enediol unit analogues are built using all combinations of the surface minima. Such a "build-up" procedure, which is very fast, is able to reproduce 75% of the minima obtained from a full dihedral angle exploration of the conformational space. The cis-enediol unit minima are compared with the corresponding di- and tripeptide minima; all peptide minima can be closely matched by a cis-enediol unit minimum of low energy (less than 2.2 kcal/mol above the lowest energy conformer). However, there are low energy minima of the cis-enediol unit that have no corresponding minima in peptides. The results are shown to depend strongly on the chirality of the analogues. The ability of each of the stereoisomers to mimic natural peptides, evaluated by the present approach, is correlated with its experimental activity in a renin inhibition assay.     

Calibration of force-field dependency in free energy landscapes of peptide conformations by quantum chemical calculations

The free energy landscapes of peptide conformations were calibrated by ab initio quantum chemical calculations, after the enhanced conformational diversity search using the multicanonical molecular dynamics simulations. Three different potentials of mean force for an isolated dipeptide were individually obtained by the multicanonical molecular dynamics simulations using the conventional force fields, AMBER parm94, AMBER parm96, and CHARMm22. Each potential of mean force was then calibrated based upon the umbrella sampling algorithm from the adiabatic energy map that was calculated separately by the ab initio molecular orbital method, and all of the calibrated potentials of mean force coincided well. The calibration method was also applied to the simulations of a peptide dimer in explicit water models, and it was shown that the calibrated free energy landscapes did not depend on the force field used in the classical simulations, as far as the conformational space was sampled well. The current calibration method fuses the classical free energy calculation with the quantum chemical calculation, and it should generally make simulations for biomolecular systems much more reliable when combining with enhanced conformational sampling.     

A CGenFF-based force field for simulations of peptoids with both cis and trans peptide bonds

Peptoids, or poly-n-substituted glycines, are peptide-like polymers composed of a flexible backbone decorated with diverse chemical side chains. Peptoids can form a variety of self-assembling structures based on the type and sequence of the side chains attached to their backbones. All-atom molecular dynamics simulations have been useful in predicting the conformational structures of proteins and will be valuable tools for identifying combinations of peptoid side chains that may form interesting folded structures. However, peptoid models must address a major degree of freedom not common in proteins – the cis/trans isomerization of the peptide bond. This work presents CHARMM general force field (CGenFF) parameters developed to accurately represent peptoid conformational behavior, with an emphasis on a correct representation of both the cis and trans isomers of the peptoid backbone. These parameters are validated against experimental and quantum mechanics data and used to simulate three peptoid side chains in explicitly solvated systems. © 2019 Wiley Periodicals, Inc.     

Peptide conformation in gas phase probed by collision-induced dissociation and its correlation to conformation in condensed phases

A kinetic peptide fragmentation model for quantitative prediction of peptide CID spectra in an ion trap mass spectrometer has been reported recently. When applying the model to predict the CID spectra of large peptides, it was often found that the predicted spectra differed significantly from their experimental spectra, presumably due to noncovalent interactions in these large polypeptides, which are not considered in the fragmentation model. As a result, site-specific quantitative information correlated to the secondary/tertiary structure of an ionized peptide may be extracted from its CID spectrum. To extract this information, the kinetic peptide fragmentation model was modified by incorporating conformation-related parameters. These parameters are optimized for best fit between the predicted and the experimental spectrum. A conformational stability map is then generated from these conformation-related parameters. Analysis of a few bioactive alpha-helical peptides including melittin, glucagon and neuropeptide Y by this technique demonstrated that their stability maps in the gas phase correlate strongly to their secondary structures in the condensed phases.     

Conformational analysis of [Met5]-enkephalin: Solvation and ionization considerations

[Met5]-Enkephalin has the sequence Tyr-Gly-Gly-Phe-Met. Only the extended conformation of the peptide has been observed by X-ray crystallography. Nuclear magnetic resonance spectroscopy supports the presence of a turn at Gly 3 and Phe 4 in dimethyl sulfoxide. In this study, the peptide conformational states and thermodynamic properties are understood in terms of ionization state and solvent environment. In the calculation, final conformations obtained from multiple independent Monte Carlo simulated annealing conformational searches are starting points for molecular dynamics simulations. In an aqueous environment given by the use of solvation free energy and the zwitterionic state, dominant structural motifs computed are G-P Type II bend, G-G Type II bend, and G-G Type I bend motifs, in order of increasing free energy. In the calculation of the peptide with neutral N- and C-termini and solvation free energy, the extended conformer dominates (by at least a factor of 2.5), and the conformation of another low free energy conformer superimposes well on the pharmacophoric groups of morphine. Neutralization of charge and solvation induce and stabilize the extended conformation, respectively. A mechanism of inter-conversion between the extended conformer and three bent conformers is supported by /-scatter plots, and by the conformer relative free energies. An estimate of the entropy change of receptor unbinding is 8.3 cal K-1 mol-1, which gives rise to a -2.5 kcal/mol entropy contribution to the free energy of unbinding at 25 °C. The conformational analysis methodology described here should be useful in studies on short peptides and flexible protein surface loops that have important biological implications.     

The influence of simulation conditions in molecular dynamics investigations of model β-sheet peptides

The influence of simulation methods, cutoff based and particle mesh Ewald (PME) on the accuracy by which experimentally derived nuclear Overhauser effect (NOE) data are reproduced, has been investigated using 500-ns-long molecular dynamics simulations on a model -sheet peptide in explicit solvent. The structural and conformational features under the different conditions were evaluated in terms of flexibility, secondary structure content, hydrogen-bonding pattern and percent of native contacts as a function of time. It was found that the different simulation methods strongly influence the dynamics of the peptide, confirming previous observations based on ideal peptide models simulated for much shorter times. Moreover, the results of our simulations prove once more that it is necessary to reach extremely long time scales to obtain enough statistics to accurately reproduce experimental NOE restraints even in the case of the PME method, despite its tendency to the stabilization of conformations which are structurally closely related to the ones derived through experiment. Possible implications regarding the stabilization and folding mechanisms, together with their relationship to the experimental study of peptide models, are discussed.     

Hamiltonian replica‐exchange simulations with adaptive biasing of peptide backbone and side chain dihedral angles

A Hamiltonian Replica‐Exchange Molecular Dynamics (REMD) simulation method has been developed that employs a two‐dimensional backbone and one‐dimensional side chain biasing potential specifically to promote conformational transitions in peptides. To exploit the replica framework optimally, the level of the biasing potential in each replica was appropriately adapted during the simulations. This resulted in both high exchange rates between neighboring replicas and improved occupancy/flow of all conformers in each replica. The performance of the approach was tested on several peptide and protein systems and compared with regular MD simulations and previous REMD studies. Improved sampling of relevant conformational states was observed for unrestrained protein and peptide folding simulations as well as for refinement of a loop structure with restricted mobility of loop flanking protein regions. © 2013 Wiley Periodicals, Inc.     

A computational investigation and the conformational analysis of dimers, anions, cations, and zwitterions of L-phenylalanine

The structure and stability of various conformations of L-phenylalanine (PheN) and its zwitterions (PheZ), along with their ionized counterparts, cation (PheC) and anion (PheA), generated by adding and removing a proton respectively, have been investigated using first principle calculations in vacuum and in solution. This is followed by an extensive study on various possible dimer (PheD) conformations, which are noncovalently bound units without a peptide bond. This study results in 52, 31, 12, 9, and 11 minimum energy structures on the potential energy surfaces of PheD, PheN, PheC, PheA, and PheZ, respectively. Several important nonbonded interactions such as hydrogen bonds, NH-π, CH-π, OH-π, and π-π interactions, which impart stability to the monomeric and dimeric units, have been analyzed. The capability and strength of the nonbonded interactions drastically changing the conformational orientations of monomeric units has been illustrated.     

NMR spectra of carotenoporphyrins. Computer-assisted conformational analysis

A computer-assisted method of conformational analysis for porphyrin molecules bearing flexible side-chains has been developed. The method utilizes the ring current-induced chemical shift changes of the side-chain protons which arise from the porphyrin macrocycle and any attached aryl rings. The treatment has been applied to a series of carotenoporphyrin molecules, which are important as models for a variety of photophysical processes in biological systems. Chemical shift data of sufficient accuracy for the conformational analysis were obtained from 500 MHz NMR experiments. The conformations of the carotenoporphyrins varied from extended ones with the carotenoid well away from the porphyrin ring to tightly folded species, depending on molecular constitution. The analytical method can be extended to other porphyrin-based systems.