Backbone N-Amination Promotes the Folding of ß-Hairpin Peptides via a Network of Hydrogen Bonds.

Molecular dynamics (MD) simulations have been used to characterize the effects of backbone N-amination of residues in a model ß-hairpin peptide. This modification is of considerable interest as N-aminated peptides have been shown to inhibit amyloid-type aggregation. Six derivatives of the ß-hairpin peptide, which contain one, two, or four N-aminated residues, have been studied. For each peptide 100 ns MD simulations starting from the folded ß-hairpin structure were performed. The effects of the N-amination prove to be very sequence dependent. N-Amination of a residue involved in interstrand hydrogen bonding (Val3) leads to unfolding of the ß-hairpin, whereas N-amination of a residue toward the C-terminus (Leu11) gives fraying at the termini of the peptide. In the other derivatives the peptide remains folded, with increasing levels of N-amination reducing the right-handed twist of the ß-hairpin and favoring population of a type II' rather than a type I' ß-turn. MD simulations (100 ns) have also been run for each peptide starting from an unfolded extended chain. Here, the peptide with four N-aminated residues shows the most folding into the ß-hairpin (34%). Analysis of the simulations shows that N-amination favors the population of ß (φ, ψ) conformations by the preceding residue due to, at least in part, a network of weak NH2(i)-CO(i) and NH2(i)-CO(i-2) hydrogen bonds. It also leads to a reduction of misfolding because of changes in the hydrogen-bonding potential. Both of these features help funnel the peptide to the folded ß-hairpin structure. The conformational insights provided through this work give a firm foundation for the design of N-aminated peptide inhibitors for modulating protein-protein interactions and aggregation.

Pharmacokinetics of carprofen in anaesthetized pigs: a preliminary study.

OBJECTIVE: To investigate the pharmacokinetics of carprofen after a single intravenous (IV) dose and multiple oral doses administered to pigs undergoing electroporation of the pancreas. STUDY DESIGN: Prospective experimental study. ANIMALS: A group of eight female pigs weighing 31.74 ± 2.24 kg (mean ± standard deviation). METHODS: Carprofen 4 mg kg-1 was administered IV after placement of a central venous catheter during general anaesthesia with isoflurane. Blood samples were collected 30 seconds before and 5, 10, 20, 30 and 60 minutes and 2, 4, 6, 8, 12 and 24 hours after carprofen administration. Subsequently, the same dose of carprofen was administered orally, daily, for 6 consecutive days and blood collected at 36, 48, 60, 72, 96, 120, 144 and 168 hours after initial carprofen administration. Plasma was analysed using liquid chromatography with mass spectrometry. Standard pharmacokinetic parameters were calculated by compartmental analysis of plasma concentration-time curves. Data are presented as mean ± standard error. RESULTS: The initial plasma concentration of IV carprofen was estimated at 54.57 ± 3.92 µg mL-1 and decreased to 8.26 ± 1.07 µg mL-1 24 hours later. The plasma elimination curve showed a bi-exponential decline: a rapid distribution phase with a distribution half-life of 0.21 ± 0.03 hours and a slower elimination phase with an elimination half-life of 17.31 ± 3.78 hours. The calculated pharmacokinetic parameters were as follows: the area under the plasma concentration-time curve was 357.3 ± 16.73 µg mL-1 hour, volume of distribution was 0.28 ± 0.07 L kg-1 and plasma clearance rate was 0.19 ± 0.009 mL minute-1 kg-1. The plasma concentration of carprofen, administered orally from days 2 to 7, varied from 9.03 ± 1.87 to 11.49 ± 2.15 µg mL-1. CONCLUSIONS AND CLINICAL RELEVANCE: Carprofen can be regarded as a long-acting non-steroidal anti-inflammatory drug in pigs.

Conformational Properties of the Chemotherapeutic Drug Analogue Epothilone A: How to Model a Flexible Protein Ligand Using Scarcely Available Experimental Data.

Epothilones are among the most potent chemotherapeutic drugs used for the treatment of cancer. Epothilone A (EpoA), a natural product, is a macrocyclic molecule containing 34 non-hydrogen atoms and a thiazole side chain. NMR studies of EpoA in aqueous solution, unbound as well as bound to αß-tubulin, and unbound in dimethyl sulfoxide (DMSO) solution have delivered sets of nuclear Overhauser effect (NOE) atom-atom distance bounds, but no structures based on NMR data are present in structural data banks. X-ray diffraction of crystals has provided structures of EpoA unbound and bound to αß-tubulin. Since both crystal structures derived from X-ray diffraction intensities do not completely satisfy the three available sets of NOE distance bounds for EpoA, molecular dynamics (MD) simulations have been employed to obtain conformational ensembles in aqueous and in DMSO solution that are compatible with the respective NOE data. It was found that EpoA displays a larger conformational variability in DMSO than in water and the two conformational ensembles show little overlap. Yet, they both provide conformational scaffolds that are energetically accessible at physiological temperature and pressure.

Detection of tetracycline and other antimicrobial residues in milk from cows with clinical mastitis treated by combination therapy.

The purpose of this study was to determine tetracycline and other antimicrobial residues in milk from dairy cows treated for clinical mastitis. Data on cow health and drugs used were obtained from the farm veterinarians. A milk sample from each affected udder quarter of each cow was taken for bacterial identification before treatment. All 35 cows in the study were treated with an intramammary product containing tetracycline, neomycin, bacitracin, and prednisolone (tetracycline injector) and also simultaneously with other drugs via the intramammary or parenteral route. The withhold period of the 'tetracycline product' was eight milkings but, in all cases, longer withhold periods were prescribed due to off-label application of additional combinations of drugs. Milk samples from treated udder quarters, taken two milkings before and two after the prescribed withhold period, were analysed for antimicrobial residues. Additionally, milk samples were taken from untreated healthy udder quarters to check if any crossover of drugs had occurred. Three screening tests were used for antimicrobial detection. In 15 (42·9%) cows the milk samples from the treated infected quarters contained tetracycline residues above the maximum residue limit (MRL) after the prescribed withhold period and, in two cases (5·7%) the same was true for neomycin and cefquinome residues. Cephalexin above the MRL was detected in only one case (2·9%). Beta-lactams did not exceed the MRL after the prescribed withhold period. Antimicrobial residues were not detected in milk samples from untreated quarters. Cow condition including parity, decreased milk production and severity of mastitis significantly influenced the excretion of antimicrobials in milk (P ≤ 0·05). No significant differences were found between the tetracycline positive and tetracycline negative cows regarding the bacteria involved, combination antimicrobial therapy, or treatment interval and duration.

A molecular dynamics simulation investigation of the relative stability of the cyclic peptide octreotide and its deprotonated and its (CF3)-Trp substituted analogs in different solvents.

The cyclic octa-peptide octreotide and its derivatives are used as diagnostics and therapeutics in relation to particular types of cancers. This led to investigations of their conformational properties using spectroscopic, NMR and CD, methods. A CF3-substituted derivative, that was designed to stabilize the dominant octreotide conformer responsible for receptor binding, turned out to have a lower affinity. The obtained spectroscopic data were interpreted as to show an increased flexibility of the CF3 derivative compared to the unsubstituted octreotide, which could then explain the lower affinity. In this article, we use MD simulation without and with time-averaged NOE distance and time-averaged local-elevation 3J-coupling restraining representing experimental NMR data to determine the conformational properties of the different peptides in the different solvents for which experimental data are available, that are compatible with the NOE atom-atom distance bounds and the 3JHNHα-couplings as derived from the NMR measurements. The conformational ensembles show that the CF3 substitution in combination with the change of solvent from water to methanol leads to a decrease in flexibility and a shift in the populations of the dominant conformers that are compatible with the experimental data.

Investigation of the structural preference and flexibility of the loop residues in amyloid fibrils of the HET-s prion.

The structural variability of a 16-residue loop (residues 246-261) which is in part disordered and connects two layers of the ß-solenoid formed by the prion-form of HET-s and its prion domain HET-s(218-289) is investigated using molecular dynamics computer simulation. A system of three HET-s(218-289) molecules in a ß-sheet structure as in the fibril is simulated in aqueous solution. The trajectory structures appear to be consistent with the Cα chemical shift data obtained. In order to delineate the influence of the ß-sheet core of the fibril upon the structural variability of the loop, the latter is also simulated without the ß-sheet core, but with its N- and C-terminal residues restrained at their positions in the fibril. The analysis of the trajectories shows that the structural variability of the loop is restricted by the ß-sheet core, least at its N-terminal end and most in the middle of the trimer.

Does Preoperative Antimicrobial Prophylaxis Influence the Diagnostic Potential of Periprosthetic Tissues in Hip or Knee Infections?

BACKGROUND: Undiagnosed low-grade prosthetic joint infections (PJI) are recognized as an important reason for early failure of presumably aseptic revisions. Preoperatively administered antimicrobial prophylaxis reduces the incidence of PJI but it may reduce the sensitivity of microbiologic periprosthetic tissue cultures and consequently increase the incidence of undiagnosed septic prosthetic joint failures, which can lead to catastrophic serial revisions. QUESTIONS/PURPOSES: We wished to determine whether administration of preoperative antibiotics decreases the likelihood of diagnosing PJI in patients undergoing revision hip or knee arthroplasty in whom infection is suspected. METHODS: We prospectively enrolled and evaluated 40 patients (29 with THAs and 11 with TKAs) who met the following inclusion criteria: older than 18 years, with suspected PJI of unknown cause, undergoing surgical revision. After arthrotomy, three tissue samples were obtained for microbiologic analysis and diagnosis, and antimicrobial prophylaxis (cefazolin 2 g intravenously) then was administered. Later during the procedure, but before débridement and irrigation, the second set of three tissue samples was obtained from the same surgical area and was cultured. Tissue concentration of prophylactic antibiotic was verified with the second set of samples. A positive culture result was defined as one or more positive cultures (growth on agar at or before 14 days). We then compared the yield on the microbiologic cultures obtained before administration of antibiotics with the yield on the cultures obtained after antibiotics were administered. An a priori analysis was performed; with the numbers available, we had 98% power to detect a difference in diagnostic sensitivity of 33%. RESULTS: With the numbers available, we found no difference in the likelihood that an infection would be diagnosed between the samples obtained before and after administration of antimicrobial prophylaxis (odds ratio [OR] for positive microbial culture = 0.99; 95% CI, 0.40-2.48; p = 0.99). All measured tissue concentrations of cefazolin were greater than the minimum inhibitory concentration, therefore we found that antibiotic prophylaxis was adequate at the time of second-set tissue specimen recovery. CONCLUSIONS: Results from this small, prospective series suggest that preoperative antimicrobial prophylaxis may be administered safely even in patients undergoing revision hip or knee arthroplasty in which microbiologic sampling is planned without compromising the diagnostic sensitivity of tissue sample cultures. However, before applying our results more generally, our findings need to be confirmed in larger, multicenter studies that would allow evaluation by sex, procedure, bacteriology, and other potentially important factors. LEVEL OF EVIDENCE: Level I, diagnostic study.

Deriving Structural Information from Experimentally Measured Data on Biomolecules.

During the past half century, the number and accuracy of experimental techniques that can deliver values of observables for biomolecular systems have been steadily increasing. The conversion of a measured value Qexp of an observable quantity Q into structural information is, however, a task beset with theoretical and practical problems: 1) insufficient or inaccurate values of Qexp , 2) inaccuracies in the function Q(r→) used to relate the quantity Q to structure r→ , 3) how to account for the averaging inherent in the measurement of Qexp , 4) how to handle the possible multiple-valuedness of the inverse r→(Q) of the function Q(r→) , to mention a few. These apply to a variety of observable quantities Q and measurement techniques such as X-ray and neutron diffraction, small-angle and wide-angle X-ray scattering, free-electron laser imaging, cryo-electron microscopy, nuclear magnetic resonance, electron paramagnetic resonance, infrared and Raman spectroscopy, circular dichroism, Förster resonance energy transfer, atomic force microscopy and ion-mobility mass spectrometry. The process of deriving structural information from measured data is reviewed with an eye to non-experts and newcomers in the field using examples from the literature of the effect of the various choices and approximations involved in the process. A list of choices to be avoided is provided.

Substitution of proline32 by α-methylproline preorganizes ß2-microglobulin for oligomerization but not for aggregation into amyloids.

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human ß2-microglobulin (ß2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a ß2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]ß2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type ß2m, [MePro32]ß2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type ß2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]ß2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial ß2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of ß2m amyloid structures.

Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Structure and conformational dynamics of the domain 5 RNA hairpin of a bacterial group II intron revealed by solution nuclear magnetic resonance and molecular dynamics simulations.

Nuclear magnetic resonance (NMR) nuclear Overhauser enhancement (NOE) data obtained for a 35-nucleotide RNA segment of a bacterial group II intron indicate a helical hairpin structure in which three parts, a terminal pentaloop, a bulge, and a G-A mismatch, display no Watson-Crick base pairing. The 668 NOE upper distance bounds for atom pairs are insufficient to uniquely determine the conformation of these segments. Therefore, molecular dynamics simulations including time-averaged distance restraints have been used to obtain a conformational ensemble compatible with the observed NMR data. The ensemble shows alternating hydrogen bonding patterns for the mentioned segments. In particular, in the pentaloop and in the bulge, the hydrogen bonding networks correspond to distinct conformational clusters that could not be captured by using conventional single-structure refinement techniques. This implies that, to obtain a realistic picture of the conformational ensemble of such flexible biomolecules, it is necessary to properly account for the conformational variability in the structure refinement of RNA fragments.

Multi-resolution simulation of biomolecular systems: a review of methodological issues.

Theoretical-computational modeling with an eye to explaining experimental observations in regard to a particular chemical phenomenon or process requires choices concerning essential degrees of freedom and types of interactions and the generation of a Boltzmann ensemble or trajectories of configurations. Depending on the degrees of freedom that are essential to the process of interest, for example, electronic or nuclear versus atomic, molecular or supra-molecular, quantum- or classical-mechanical equations of motion are to be used. In multi-resolution simulation, various levels of resolution, for example, electronic, atomic, supra-atomic or supra-molecular, are combined in one model. This allows an enhancement of the computational efficiency, while maintaining sufficient detail with respect to particular degrees of freedom. The basic challenges and choices with respect to multi-resolution modeling are reviewed and as an illustration the differential catalytic properties of two enzymes with similar folds but different substrates with respect to these substrates are explored using multi-resolution simulation at the electronic, atomic and supra-molecular levels of resolution.

Influence of 63Ser phosphorylation and dephosphorylation on the structure of the stathmin helical nucleation sequence: a molecular dynamics study.

Phosphorylation is an important mechanism regulating protein-protein interactions involving intrinsically disordered protein regions. Stathmin, an archetypical example of an intrinsically disordered protein, is a key regulator of microtubule dynamics in which phosphorylation of 63Ser within the helical nucleation sequence strongly down-regulates the tubulin binding and microtubule destabilizing activities of the protein. Experimental studies on a peptide encompassing the 19-residue helical nucleation sequence of stathmin (residues 55-73) indicate that phosphorylation of 63Ser destabilizes the peptide's secondary structure by disrupting the salt bridges supporting its helical conformation. In order to investigate this hypothesis at atomic resolution, we performed molecular dynamics simulations of nonphosphorylated and phosphorylated stathmin-[55-73] at room temperature and pressure, neutral pH, and explicit solvation using the recently released GROMOS force field 54A7. In the simulations of nonphosphorylated stathmin-[55-73] emerged salt bridges associated with helical configurations. In the simulations of 63Ser phosphorylated stathmin-[55-73] these configurations dispersed and were replaced by a proliferation of salt bridges yielding disordered configurations. The transformation of the salt bridges was accompanied by emergence of numerous interactions between main and side chains, involving notably the oxygen atoms of the phosphorylated 63Ser. The loss of helical structure induced by phosphorylation is reversible, however, as a final simulation showed. The results extend the hypothesis of salt bridge derangement suggested by experimental observations of the stathmin nucleation sequence, providing new insights into regulation of intrinsically disordered protein systems mediated by phosphorylation.

Assessment of enveloping distribution sampling to calculate relative free enthalpies of binding for eight netropsin-DNA duplex complexes in aqueous solution.

The performance of enveloping distribution sampling (EDS) simulations to estimate free enthalpy differences associated with seven alchemical transformations of A-T into G-C base pairs at the netropsin binding site in the minor groove of a 13-base pair DNA duplex in aqueous solution is evaluated. It is demonstrated that sufficient sampling can be achieved with a two-state EDS Hamiltonian even for large perturbations such as the simultaneous transformation of up to three A-T into three G-C base pairs. The two parameters required to define the EDS reference state Hamiltonian are obtained automatically using a modified version of a scheme presented in earlier work. The sensitivity of the configurational sampling to a variation of these parameters is investigated in detail. Although for relatively small perturbations, that is, one base pair, the free enthalpy estimate depends only weakly on the EDS parameters, the sensitivity is stronger for the largest perturbation. Yet, EDS offers various convenient measures to evaluate the degree of sampling and thus the reliability of the free enthalpy estimate and appears to be an efficient alternative to the conventional thermodynamic integration methodology to obtain free energy differences for molecular systems.

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea.

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species-P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle-have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

Biomolecular structure refinement using the GROMOS simulation software.

For the understanding of cellular processes the molecular structure of biomolecules has to be accurately determined. Initial models can be significantly improved by structure refinement techniques. Here, we present the refinement methods and analysis techniques implemented in the GROMOS software for biomolecular simulation. The methodology and some implementation details of the computation of NMR NOE data, (3)J-couplings and residual dipolar couplings, X-ray scattering intensities from crystals and solutions and neutron scattering intensities used in GROMOS is described and refinement strategies and concepts are discussed using example applications. The GROMOS software allows structure refinement combining different types of experimental data with different types of restraining functions, while using a variety of methods to enhance conformational searching and sampling and the thermodynamically calibrated GROMOS force field for biomolecular simulation.

Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

Molecular simulation as an aid to experimentalists.

Computer-based molecular simulation techniques are increasingly used to interpret experimental data on biomolecular systems at an atomic level. Direct comparison between experiment and simulation is, however, seldom straightforward. The available experimental data are limited in scope and generally correspond to averages over both time and space. A critical analysis of the various factors that may influence the apparent degree of agreement between the results of simulations and experimentally measured quantities is presented and illustrated using examples from recent literature.

Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1.

The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16-31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 phi torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular alpha-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured (3)J(H(N)-H(Calpha))-coupling constants, indicating that different conformers of GCN4p16-31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and (3)J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and (3)J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16-31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16-31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.

Conformational editing of intrinsically disordered protein by α-methylation.

Intrinsically disordered proteins (IDPs) constitute a large portion of "Dark Proteome" - difficult to characterize or yet to be discovered protein structures. Here we used conformationally constrained α-methylated amino acids to bias the conformational ensemble in the free unstructured activation domain of transcriptional coactivator ACTR. Different sites and patterns of substitutions were enabled by chemical protein synthesis and led to distinct populations of α-helices. A specific substitution pattern resulted in a substantially higher binding affinity to nuclear coactivator binding domain (NCBD) of CREB-binding protein, a natural binding partner of ACTR. The first X-ray structure of the modified ACTR domain - NCBD complex visualized a unique conformation of ACTR and confirmed that the key α-methylated amino acids are localized within α-helices in the bound state. This study demonstrates a strategy for characterization of individual conformational states of IDPs.