Conformational change in unsolvated Trp-cage protein probed by fluorescence

We report the first direct measurements of the unfolding of a protein, Trp-cage, in the gas phase using laser-induced fluorescence of protein ions in a heated quadrupole ion trap. The changes in enthalpy and entropy associated with the observed conformational change are obtained by fitting a two-state model of protein unfolding to the fluorescence intensities plotted versus temperature. The enthalpy and entropy changes for the 2+ and 3+ charge states are greater than the values measured in solution and depend on charge state.     

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.     

A direct comparison of protein structure in the gas and solution phase: the Trp-cage

Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location, there is significant difference in the structures, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts in the gas phase. The structure of the salt bridge in the gas phase is also much more stable than in the (experimental) solution structure.     

Influence of water-protein hydrogen bonding on the stability of Trp-cage miniprotein. A comparison between the TIP3P and TIP4P-Ew water models

We report extensive replica exchange molecular dynamics (REMD) simulations on the folding/unfolding equilibrium of Trp-cage miniprotein using the Amber ff99SB all atom forcefield and TIP3P and TIP4P-Ew explicit water solvent models. REMD simulation-lengths in the 500 ns to the microsecond regime per replica are required to adequately sample the folding/unfolding equilibrium. We observe that this equilibrium is significantly affected by the choice of the water model. Compared with experimental data, simulations using the TIP3P solvent describe the stability of the Trp-cage quite realistically, providing a melting point which is just a few Kelvins above the experimental transition temperature of 317 K. The TIP4P-Ew model shifts the equilibrium towards the unfolded state and lowers the free energy of unfolding by about 3 kJ mol(-1) at 280 K, demonstrating the need to fine-tune the protein-forcefield depending on the chosen water model. We report evidence that the main difference between the two water models is mostly due to the different solvation of polar groups of the peptide. The unfolded state of the Trp-cage is stabilized by an increasing number of hydrogen bonds, destabilizing the α-helical part of the molecule and opening the R-D salt bridge. By reweighting the strength of solvent-peptide hydrogen bonds by adding a hydrogen bond square well potential, we can fully recover the effect of the different water models and estimate the shift in population as due to a difference in hydrogen bond-strength of about 0.4 kJ mol(-1) per hydrogen bond.     

Fluorescence quenching induced by conformational fluctuations in unsolvated polypeptides

Time-resolved measurements were conducted to relate the fluorescence lifetimes of dye-derivatized polypeptides to local conformational dynamics in trapped, unsolvated peptide ions. This research was performed to better understand the intramolecular interactions leading to the observed increase of fluorescence quenching with temperature and, in particular, how this quenching is related to conformational fluctuations. Dye-derivatized polyproline ions, Dye-[Pro] n -Arg (+)-Trp, are formed by electrospray ionization and trapped in a variable-temperature quadrupole ion trap where they are exposed to a pulsed laser which excites fluorescence. Lifetime data exhibit fluorescence quenching as a result of an interaction between the dye and tryptophan (Trp) side chain. This result is consistent with solution measurements performed for comparison. The lifetime temperature dependence is closely fit over the range 150-463 K by an Arrhenius model of the ensemble averaged quenching rate, k q. Model fits of the measured lifetimes yield a frequency prefactor of approximately 10 (11) s (-1) for k q characteristic of collective motions of the side chains identified in molecular dynamics (MD) simulations. The data fits also yield activation barriers of approximately 0.3 eV, which are comparable to intramolecular electrostatic interactions calculated between the unshielded charge on the Arg residue and the dye. As a result, the quenching rate appears to be determined by the rate of conformational fluctuations and not by the rate of a specific quenching mechanism. The peptide sequence of Dye-Trp-[Pro] n -Arg (+) was also studied and identified a dependence of the quenching rate on the electrostatic field in the vicinity of the dye, Trp pair. Molecular dynamics simulations were performed over the range of experimental measurements to study trajectories relevant to the quenching interaction. The MD simulations indicate that as the temperature is increased, conformational fluctuations in the presence of strong electrostatic fields of the charged Arg (+) residue can result in both (a) an increased number of dye and Trp separations <8 A and (b) increased exothermicity for electron transfer reactions between the dye and Trp. Consequently, the MD simulations are consistent with increased fluorescence quenching with temperature resulting from the occurrence of conformers having specific positions of the dye, Trp, and Arg (+). As a result, the fluorescence lifetime provides a local probe of conformational fluctuations averaged over the ion ensemble.     

Do Charge State Signatures Guarantee Protein Conformations?

The extent to which proteins in the gas phase retain their condensed-phase structure is a hotly debated issue. Closely related to this is the degree to which the observed charge state reflects protein conformation. Evidence from electron capture dissociation, hydrogen/deuterium exchange, ion mobility, and molecular dynamics shows clearly that there is often a strong correlation between the degree of folding and charge state, with the most compact conformations observed for the lowest charge states. In this article, we address recent controversies surrounding the relationship between charge states and folding, focussing also on the manipulation of charge in solution and its effect on conformation. 'Supercharging' reagents that have been used to effect change in charge state can promote unfolding in the electrospray droplet. However for several protein complexes, supercharging does not appear to perturb the structure in that unfolding is not detected. Consequently, a higher charge state does not necessarily imply unfolding. Whilst the effect of charge manipulation on conformation remains controversial, there is strong evidence that a folded, compact state of a protein can survive in the gas phase, at least on a millisecond timescale. The exact nature of the side-chain packing and secondary structural elements in these compact states, however, remains elusive and prompts further research.     

Preferred conformation of the glycosidic linkage of methyl-beta-mannose

The conformational preference of the glycosidic linkage of methyl-beta-mannose was studied in the gas phase and in aqueous solution by ab initio calculations, and by molecular dynamics (MD) and Car-Parrinello molecular dynamics (CPMD) simulations. MD simulations were performed with various water potential functions to study the impact of the chosen water potential on the predicted conformational preference of the glycosidic linkage of the carbohydrate in solution. This study shows that the trans (t) orientation of the glycosidic linkage of methyl-beta-mannose is preferred over its gauche clockwise (g+) orientation in solution. CPMD simulations clearly indicate that this preference is due to intermolecular hydrogen bonding with surrounding water molecules, whereas no such information could be demonstrated by MD simulations. This study demonstrates the importance of ab initio molecular dynamics simulations in studying the structural properties of carbohydrate-water interactions.     

All-atom Molecular Dynamics Simulationsand NMR Spectroscopy Study on Interactions and Structures in N-Glycylglycine Aqueous Solution

All-atom molecular dynamics (MD) simulation and the NMR spectra are used to investi-gate the interactions in N-glycylglycine aqueous solution. Different types of atoms exhibit different capability in forming hydrogen bonds by the radial distribution function analysis. Some typical dominant aggregates are found in different types of hydrogen bonds by the statistical hydrogen-bonding network. Moreover, temperature-dependent NMR are used to compare with the results of the MD simulations. The chemical shifts of the three hydrogen atoms all decrease with the temperature increasing which reveals that the hydrogen bonds are dominant in the glycylglycine aqueous solution. And the NMR results show agreement with the MD simulations. All-atom MD simulations and NMR spectra are successful in revealing the structures and interactions in the N-glycylglycine-water mixtures.     

Interpreting protein structural dynamics from NMR chemical shifts

In this investigation, semiempirical NMR chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (MD) simulations of proteins. MD-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static X-ray structures. Improved chemical shift predictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations within conformational basins. Improved chemical shift predictions also result from discrete changes to conformations observed in X-ray structures, which may result from crystal contacts, and are not always reflective of conformational dynamics in solution. Chemical shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, and averaged (1)H chemical shifts are particularly sensitive reporters of fluctuations in aromatic ring positions and geometries of hydrogen bonds. In addition, poor predictions of MD-averaged chemical shifts can identify spurious conformations and motions observed in MD simulations that may result from force field deficiencies or insufficient sampling and can also suggest subsets of conformational space that are more consistent with experimental data. These results suggest that the analysis of dynamically averaged NMR chemical shifts from MD simulations can serve as a powerful approach for characterizing protein motions in atomistic detail.     

Study on the conformational equilibrium of the alanine dipeptide in water solution by using the averaged solvent electrostatic potential from molecular dynamics methodology

The ASEP/MD method has been employed for studying the solvent effect on the conformational equilibrium of the alanine dipeptide in water solution. MP2 and density functional theory (DFT) levels of theory were used and results were compared. While in gas phase cyclic structures showing intramolecular hydrogen bonds were found to be the most stable, the stability order is reversed in water solution. Intermolecular interaction with the solvent causes the predominance of extended structures as the stabilizing contacts dipeptide-water are favoured. Free-energy differences in solution were calculated and PPII, α(R), and C5 conformers were identified as the most stable at MP2 level. Experimental data from Raman and IR techniques show discrepancies about the relative abundance of α(R) y C5, our results support the Raman data. The DFT level of theory agrees with MP2 in the location and stability of PPII and α(R) forms but fails in the location of C5. MP2 results suggest the possibility of finding traces of C7eq conformer in water solution, in agreement with recent experiments.     

Solvation properties of N-acetyl-β-glucosamine: molecular dynamics study incorporating electrostatic polarization

N-Acetyl-β-glucosamine (NAG) is an important moiety of glycoproteins and is involved in many biological functions. However, conformational and dynamical properties of NAG molecules in aqueous solution, the most common biological environment, remain ambiguous due to limitations of experimental methods. Increasing efforts are made to probe structural properties of NAG and NAG-containing macromolecules, like peptidoglycans and polymeric chitin, at the atomic level using molecular dynamics simulations. In this work, we develop a polarizable carbohydrate force field for NAG and contrast simulation results of various properties using this novel force field and an analogous nonpolarizable (fixed charge) model. Aqueous solutions of NAG and its oligomers are investigated; we explore conformational properties (rotatable bond geometry), electrostatic properties (dipole moment distribution), dynamical properties (self-diffusion coefficient), hydrogen bonding (water bridge structure and dynamics), and free energy of hydration. The fixed-charge carbohydrate force field exhibits deviations from the gas phase relative rotation energy of exocyclic hydroxymethyl side chain and of chair/boat ring distortion. The polarizable force field predicts conformational properties in agreement with corresponding first-principles results. NAG-water hydrogen bonding pattern is studied through radial distribution functions (RDFs) and correlation functions. Intermolecular hydrogen bonding between solute and solvent is found to stabilize NAG solution structures while intramolecular hydrogen bonds define glycosidic linkage geometry of NAG oligomers. The electrostatic component of hydration free energy is highly dependent on force field atomic partial charges, influencing a more favorable free energy of hydration in the fixed-charge model compared to the polarizable model.     

Glycosidic linkage conformation of methyl-alpha-mannopyranoside

We study the preferred conformation of the glycosidic linkage of methyl-alpha-mannopyranoside in the gas phase and in aqueous solution. Results obtained utilizing Car-Parrinello molecular dynamics (CPMD) simulations are compared to those obtained from classical molecular dynamics (MD) simulations. We describe classical simulations performed with various water potential functions to study the impact of the chosen water potential on the predicted conformational preference of the glycosidic linkage of the carbohydrate in aqueous solution. In agreement with our recent studies, we find that results obtained with CPMD simulations differ from those obtained from classical simulations. In particular, this study shows that the trans (t) orientation of the glycosidic linkage of methyl-alpha-mannopyranoside is preferred over its gauche anticlockwise (g-) orientation in aqueous solution. CPMD simulations indicate that this preference is due to intermolecular hydrogen bonding with surrounding water molecules, whereas no such information could be demonstrated by classical MD simulations. This study emphasizes the importance of ab initio MD simulations for studying the structural properties of carbohydrates in aqueous solution.     

Molecular Dynamics Simulations of a β-Hairpin Fragment of Protein G by Means of Atom-Bond Electronegativity Equalization Method Fused into Molecular Mechanics (ABEEMσπ/MM)

There are some controversial opinions about the origin of folding β‐hairpin stability in aqueous solution. In this study, the structural and dynamic behavior of a 16‐residue β‐hairpin from B1 domain of protein G has been investigated at 280, 300, 350 and 450 K using molecular dynamics (MD) simulations by means of Atom‐Bond Electronegativity Equalization Method Fused into Molecular Mechanics i.e., ABEEMδπ/MM and the explicit ABEEM‐7P water solvent model. In addition, a 300 K simulation of one mutant having the aromatic residues substituted with alanines has been performed. The hydrophobic surface area, hydrophilic surface area and some structural properties have been used to measure the role of the hydrophobic interactions. It is found that the aromatic residues substituted with alanines have shown an evident destabilization of the structure and unfolding started after 1.5 ns. It is also found that the number of the main chain hydrogen bonds have different distributions through three different simulations. All above demonstrate that the hydrophobic interactions and the main chain hydrogen bonds play an important role in the stability of the folding structure of β‐hairpin in solution. Furthermore, through the structural analyses of the β‐hairpin structures from four temperature simulations and the comparison with other MD simulations of β‐hairpin peptides, the new ABEEMδπ force field can reproduce the structural data in good agreement with the experimental data.     

Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs–ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) – a model of a large dynamic enzyme in the µs–ms range – by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision‐induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas‐phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co‐existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.     

Effects of guanidinium ions on the conformational structure of glucose oxidase studied by electrochemistry, spectroscopy, and theoretical calculations: towards developing a chemical-induced protein conformation assay

Understanding conformation transitions of proteins in the presence of a chemical denaturant is a topic of great interest because the rich information contained in chemical unfolding is of fundamental importance for proteomic and pharmaceutical research. In this work, the conformational structure changes of glucose oxidase (GOx) induced by guanidinium ions (Gdm(+)) were studied in detail by a combination of electrochemical methods, various spectroscopic techniques including ultraviolet-visible (UV-vis) absorption, fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations with the purpose of revealing the mechanism of chemical unfolding of proteins. The results indicated that GOx underwent substantial conformational changes both at the secondary and tertiary structure levels after interacting with Gdm(+) ions. The interaction of GOx with the chemical denaturant resulted in a disturbance of the structure of the flavin prosthetic group (FAD moiety) that induced the moiety to become less exposed to solvent than that in the native protein molecule. The calculation from quantitative second-derivative infrared and CD spectra showed that Gdm(+) ions induced the conversion of α-helix to β-sheet structures. MD simulations and DFT calculations revealed that Gdm(+) ions could enter the active pocket of the GOx molecule and interact with the FAD group, leading to a significant alteration in the structural characteristics and hydrogen bond networks formed between FAD and the surrounding amino acid residues. These alterations in the conformational structure of GOx resulted in a significant decrease in the catalytic activity of the enzyme to glucose oxidation. The study essentially provides an effective way for investigating the mechanism of chemical denaturant-induced protein unfolding, and this approach can be used for assessing the effect of drug molecules on proteins.     

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, α-chymotrypsinogen A, β-lactoglobulin B, and holo α-lactalbumin, are studied on Sepharose™ HIC resins possessing assorted ligand chemistries and densities. α-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed β-lactoglobulin B and holo α-lactalbumin range from −4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2–0.9 min⁻¹. While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.     

Y220C突变体影响p53C蛋白质构象转换的分子动力学模拟

p53是迄今发现突变频率最高的一种肿瘤抑制蛋白质,突变会导致p53抑癌功能丧失并诱导癌症的发生。绝大多数的突变发生在p53的核心DNA结合区域（p53C）,其中Y220C是研究较多的一种突变体。虽然已有研究表明该突变能够降低p53C的结构稳定性,但其影响p53C构象转换的分子机制尚不清晰。本文利用分子动力学（MD）模拟方法研究了p53C突变体Y220C（p53C-Y220C）的结构变化,发现Y220C突变主要影响Y220C cluster区域（包括残基138-164和215-238）,且Y220C突变减少了Y220C cluster的β-折叠含量。进一步分析发现,Y220C突变不仅直接破坏突变氨基酸与周围氨基酸Leu145和Thr155之间的氢键,而且降低了Y220C cluster区域的折叠片S3和S8之间的氢键数量,使Y220C突变所形成的亲水性空腔变大,加速了水分子进入该蛋白质内部,并最终导致了p53C-Y220C变性。MD模拟结果揭示了Y220C突变影响p53C结构转换的分子机制,该研究对p53C-Y220C突变体高效稳定剂的筛选和设计具有重要意义。     

Combination of molecular dynamics method and 3D-RISM theory for conformational sampling of large flexible molecules in solution

We have developed an algorithm for sampling the conformational space of large flexible molecules in solution, which combines the molecular dynamics (MD) method and the three-dimensional reference interaction site model (3D-RISM) theory. The solvent-induced force acting on solute atoms was evaluated as the gradient of the solvation free energy with respect to the solute-atom coordinates. To enhance the computation speed, we have applied a multiple timestep algorithm based on the RESPA (Reversible System Propagator Algorithm) to the combined MD/3D-RISM method. By virtue of the algorithm, one can choose a longer timestep for renewing the solvent-induced force compared with that of the conformational update. To illustrate the present MD/3D-RISM simulation, we applied the method to a model of acetylacetone in aqueous solution. The multiple timestep algorithm succeeded in enhancing the computation speed by 3.4 times for this model case. Acetylacetone possesses an intramolecular hydrogen-bonding capability between the hydroxyl group and the carbonyl oxygen atom, and the molecule is significantly stabilized due to this hydrogen bond, especially in gas phase. The intramolecular hydrogen bond was kept intact during almost entire course of the MD simulation in gas phase, while in the aqueous solutions the bond is disrupted in a significant number of conformations. This result qualitatively agrees with the behavior on a free energy barrier lying upon the process for rotating a torsional degree of freedom of the hydroxyl group, where it is significantly reduced in aqueous solution by a cancellation between the electrostatic interaction and the solvation free energy.     

Helices and Sheets in vacuo

The structures and properties of unsolvated peptides large enough to possess secondary structure have been examined by experiments and simulations. Some of the factors that stabilize unsolvated helices and sheets have been identified. The charge, in particular, plays a critical role in stabilizing alpha-helices and destabilizing beta-sheets. Some helices are much more stable in vacuum than in aqueous solution. Factors like helix propensity, context, and the incorporation of specific stabilizing interactions have been examined. The helix propensities in vacuum differ from those found in solution. Studies of the hydration of unsolvated peptides can be performed one water molecule at a time. The first few water molecules only bind weakly to unsolvated peptides, and they bind much more strongly to some conformations than to others. The most favorable binding locations are not the protonation sites, but clefts or pockets where a water molecule can establish a network of hydrogen bonds. Non-covalent interactions between secondary structure elements leads to the formation of tertiary structure. Helical peptides assemble into complexes with a variety of intriguing structures. The intramolecular coupling of helices to make antiparallel coiled-coil geometries has also been investigated with model peptides.     

Stabilizing helical polyalanine peptides with negative polarity or charge: capping with cysteine

Alanine-based peptides are widely known for their propensity to form helices, whether in the gas phase or in aqueous solution. Interactions of substituent groups or peptides with the helical macrodipole may either encourage or discourage the formation or stability of a helix, depending upon the placement of these groups. We report the first study of the inductive stabilization of a number of peptides through electronegative or anionic N-terminal residue capping. Using Charmm27/CMAP equilibrium and replica-exchange (REX) molecular dynamics (MD) simulations with Generalized Born implicit solvation methods, we find that the N-terminal cysteine capping of alanine peptides strongly enhances the helicity, even allowing the helical moiety to remain at temperatures beyond the denaturing temperature. Though the overall number of hydrogen bonds is enhanced, this stabilization seems to occur indirectly through interaction with the helical macrodipole rather than as a direct result of hydrogen bonding involving the cysteine, though the nature of the hydrogen bonding changes.