Molecular insight into the interaction between IFABP and PA by using MM-PBSA and alanine scanning methods

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with alanine-scanning mutagenesis is a very important tool for rational drug design. In this study, molecular dynamics (MD) and MM-PBSA were applied to calculate the binding free energy between the rat intestinal fatty acid binding protein (IFABP) and palmitic acid (PA) to gain insight to the interaction details. Equally spaced snapshots along the trajectory were chosen to perform the binding free energy calculation, which yields a result highly consistent with experimental value with a deviation of 0.4 kcal/mol. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in IFABP to alanine and recomputing the DeltaDeltaG(binding). By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated DeltaDeltaG(binding) is below 1.5 kcal/mol for most of the alanine mutations of the noncharged residues (67% in total). To further investigate some particular mutants, three additional dynamical simulations of IFABP Arg126Ala, Arg106Ala, and Arg106Gln mutants were conducted. Recalculated binding free energies are well consistent with the experimental data. Moreover, the ambiguous role of Arg106 caused by the free energy change of the opposite sign when it is mutated to alanine and glutamine respectively is clarified both structurally and energetically. Typically, this can be attributed to the partial electrostatic compensation mainly from Arg56 and the obvious entropy gain in Arg106Ala mutant while not in Arg106Gln mutant. The presented structural model of IFABP-PA complex could be used to guide future studies.     

Vibrational Raman optical activity characterization of poly(l-proline) II helix in alanine oligopeptides

A vibrational Raman optical activity (ROA) study of a series of alanine peptides in aqueous solution is presented. The seven-alanine peptide Acetyl-OOAAAAAAAOO-Amide (OAO), recently shown by NMR and UVCD to adopt a predominantly poly(l-proline II) (PPII) helical conformation in aqueous solution, gave an ROA spectrum very similar to that of disordered poly(l-glutamic acid) which has long been considered to adopt the PPII conformation, both being dominated by a strong positive extended amide III ROA band at approximately 1319 cm-1 together with weak positive amide I ROA intensity at approximately 1675 cm-1. A series of alanine peptides Ala2-Ala6 studied in their cationic states in aqueous solution at low pH displayed ROA spectra which steadily evolved toward that of OAO with increasing chain length. As well as confirming that alanine peptides can support the PPII conformation in aqueous solution, our results also confirm the previous ROA band assignments for PPII structure, thereby reinforcing the foundation for ongoing ROA studies of unfolded and partially folded proteins.     

Peptide bond vibrational coupling

Neutral trialanine (Ala3), which is geometrically constrained to have its peptide bond at Phi and Psi angles of alpha-helix and PPII-like conformers, are studied at the B3LYP/6-31+G(d,p) level of theory to examine vibrational interactions between adjacent peptide units. Delocalization of the amide I, amide II, and amide III3 vibrations are analyzed by calculating their potential energy distributions (PED). The vibrational coupling strengths are estimated from the frequency shifts between the amide vibrations of Ala3 and the local amide bond vibrations of isotopically substituted Ala3 derivatives. Our calculations show the absence of vibrational coupling of the amide I and amide II bands in the PPII conformations. In contrast, the alpha-helical conformation shows strong coupling between the amide I vibrations due to the favorable orientation of the C=O bonds and the strong transitional dipole coupling. The amide III3 vibration shows weak coupling in both the alpha-helix and PPII conformations; this band can be treated as a local independent vibration. Our calculated results in general agree with our previous experimental UV Raman studies of a 21-residue mainly alanine-based peptide (AP).     

Conformational manifold of alpha-aminoisobutyric acid (Aib) containing alanine-based tripeptides in aqueous solution explored by vibrational spectroscopy, electronic circular dichroism spectroscopy, and molecular dynamics simulations

Replacement of the alpha-proton of an alanine residue to generate alpha-aminoisobutyric acid (Aib) in alanine-based oligopeptides favors the formation of a 3(10) helix when the length of the oligopeptide is about four to six residues. This research was aimed at experimentally identifying the structural impact of an individual Aib residue in an alanine context of short peptides in water and Aib's influence on the conformation of nearest-neighbor residues. The amide I band profile of the IR, isotropic and anisotropic Raman, and vibrational circular dichroism (VCD) spectra of Ac-Ala-Ala-Aib-OMe, Ac-Ala-Aib-Ala-OMe, and Ac-Aib-Ala-Ala-OMe were measured and analyzed in terms of different structural models by utilizing an algorithm that exploits the excitonic coupling between amide I' modes. The conformational search was guided by the respective 1H NMR and electronic circular dichroism spectra of the respective peptides, which were also recorded. From these analyses, all peptides adopted multiple conformations. Aib predominantly sampled the right-handed and left-handed 3(10)-helix region and to a minor extent the bridge region between the polyproline (PPII) and the helical regions of the Ramachandran plot. Generally, alanine showed the anticipated PPII propensity, but its conformational equilibrium was shifted towards helical conformations in Ac-Aib-Ala-Ala-OMe, indicating that Aib can induce helical conformations of neighboring residues positioned towards the C-terminal direction of the peptide. An energy landscape exploration by molecular dynamics simulations corroborated the results of the spectroscopic studies. They also revealed the dynamics and pathways of potential conformational transitions of the corresponding Aib residues.     

Ab Initio MP2 and Density Functional Theory Computational Study of AcAlaNH2 Peptide Hydration: A Bottom‐Up Approach

AcAlaNH₂?n H₂O (n=1–13) complexes have been proposed as models to account for water solvent effects on the molecular properties of N‐acetyl‐L ‐alanine amide. Ab initio computations are planned to evaluate peptide–water interactions and to provide a means for approximating relative effects of the short‐range many‐body interactions arising in real solution without introducing any external parameters intended to quantify empirical or semiempirical potential‐energy functions. The present bottom‐up approach reveals the formation of compact ring clusters of water molecules strongly bonded to peptidic polar groups throughout hydrogen bonds. The explicit coordination of water molecules around the peptide renders the fully extended (FE) and polyproline II (PPII) conformers more stable with respect to the 3₁₀ helix or γ turn. The alternance of donor and acceptor groups on both sides of the FE and PPII conformers allows for synergy and extensive H‐bonding.     

Molecular dynamics simulation study on stabilities and reactivities of NADH cytochrome B5 reductase

Binding free energies between coenzyme (FAD and NADH) and the apoenzyme of NADH-cytochrome b5 reductase (b5R) were estimated by applying the continuum Poisson-Boltzmann (PB) model to structures sampled from molecular dynamics simulations in explicit water molecules. Important residues for the enzymatic catalysis were clarified using a computational alanine scanning method. The binding free energies calculated by applying an alanine scanning method can successfully reproduce the trends of the measured steady-state enzymatic activities kcatNADH/KmNADH. Significant decreases in the binding free energy are expected when one of the four residues Arg91, Lys110, Ser127, and Thr181 is mutated into Ala. According to the results of the molecular dynamics simulation, Thr181 is considered to be one of the key residues that helps NADH to approach the isoalloxazine in FAD. Finally, we have constructed very simplified model systems and carried out density functional theory calculations using B3LYP/LANL2DZ//ROHF(or RHF)/LANL2DZ level of theory in order to elucidate a realistic and feasible mechanism of the hydride-ion transfer from NADH to FAD affected by HEME(Fe3+) as an electron acceptor. Our calculated results suggest that the electron and/or hydride-ion transfer reaction from NADH to FAD can be accelerated in the presence of HEME(Fe3+).     

UV Raman demonstrates that alpha-helical polyalanine peptides melt to polyproline II conformations

We examined the 204-nm UV Raman spectra of the peptide XAO, which was previously found by Shi et al.'s NMR study to occur in aqueous solution in a polyproline II (PPII) conformation. The UV Raman spectra of XAO are essentially identical to the spectra of small peptides such as ala(5) and to the large 21-residue predominantly Ala peptide, AP. We conclude that the non-alpha-helical conformations of these peptides are dominantly PPII. Thus, AP, which is highly alpha-helical at room temperature, melts to a PPII conformation. There is no indication of any population of intermediate disordered conformations. We continued our development of methods to relate the Ramachandran Psi-angle to the amide III band frequency. We describe a new method to estimate the Ramachandran Psi-angular distributions from amide III band line shapes measured in 204-nm UV Raman spectra. We used this method to compare the Psi-distributions in XAO, ala(5), the non-alpha-helical state of AP, and acid-denatured apomyoglobin. In addition, we estimated the Psi-angle distributions of peptide bonds which occur in non-alpha-helix and non-beta-sheet conformations in a small library of proteins.     

自由能微扰法研究系列烷基芳基磺酸盐的胶束化焓-熵补偿现象

为了研究不同结构的表面活性剂分子在水溶液中的胶束化焓-熵补偿现象, 采用自由能微扰(FEP)法计算了系列烷基芳基磺酸盐的溶剂化自由能, 并根据胶团化过程的质量作用模型讨论了相关热力学性质. 结果表明: 自由能微扰法得到的溶剂化自由能大小与用传统热力学表面张力法测定的吉布斯自由能相近, 能够用于比较不同结构的烷基芳基磺酸盐间胶束化能力; 烷基芳基磺酸盐在水溶液中的胶束化过程是自发进行的, 且存在焓-熵补偿现象, 补偿温度范围均在(302±2) K; 随着分子结构中芳环向长烷基链中间位置移动, 胶束化能力和胶束的稳定性均下降; 而随着芳环上短烷基链或长烷基链碳数的增加, 形成胶束的能力与稳定性均提高.     

Conformational preferences of proline oligopeptides

A conformational study on the terminally blocked proline oligopeptides, Ac-(Pro)(n)()-NMe(2) (n = 2-5), is carried out using the ab initio Hartree-Fock level of theory with the self-consistent reaction field method in the gas phase and in solutions (chloroform, 1-propanol, and water) to explore the preference and transition between polyproline II (PPII) and polyproline I (PPI) conformations depending on the chain length, the puckering, and the solvent. The mean differences in the free energy per proline of the up-puckered conformations relative to the down-puckered conformations for both diproline and triproline increases for the PPII-like conformations and decreases for the PPI-like conformations as the solvent polarity increases. These calculated results indicate that the PPII-like structures have preferentially all-down puckerings in solutions, whereas the PPI-like structures have partially mixed puckerings. The free energy difference per proline residue between the PPII- and PPI-like structures decreases as the proline chain becomes longer in the gas phase but increases as the proline chain becomes longer in solutions and the solvent polarity increases. In particular, our calculated results indicate that each of the proline oligopeptides can exist as an ensemble of conformations with the trans and cis peptide bonds in solutions, although the PPII-like structure with all-trans peptide bonds is dominantly preferred, which is reasonably consistent with the previously observed results. In diproline Ac-(Pro)(2)-NMe(2), the rotational barrier to the cis-to-trans isomerization for the first prolyl peptide bond increases as the solvent polarity increases, whereas the rotational barrier for the second prolyl peptide bond does not show the monotonic increase as the solvent polarity increases. When the rotational barriers for these two prolyl peptide bonds were compared, it could be deduced that the conformational transition from PPI with the cis peptide bond to PPII with the trans peptide bond is initiated at the C-terminus and proceeds to the N-terminus in water. This is consistent with the results from NMR experiments on polyproline in D(2)O but opposite to the results from enzymatic hydrolysis kinetics experiments on polyproline.     

Convergent Synthesis of Repeating Peptides (Val-X-Leu-Pro-Pro-Pro)(8) Adopting a Polyproline II Conformation

The N-terminal domain of maize gamma-zein has a repetitive structure (Val-His-Leu-Pro-Pro-Pro)(8) that has recently been shown to adopt an amphipathic polyproline II type conformation in aqueous solution. We report here the synthesis and conformational analysis of three model peptides (Val-X-Leu-Pro-Pro-Pro)(8) (X = Ala (1), Glu (2), Lys (3)). The three compounds have been synthesized in a very efficient way using a convergent solid-phase strategy. Circular dichroism shows unequivocally that the three model peptides adopt polyproline II (PPII) type conformations under a variety of experimental conditions and that neither the presence of histidine nor amphipathicity of the peptide is an absolute requirement for adopting the native conformation. These results open the door for the de novo design of compounds with PPII conformations and must be taken into account in the structure prediction of protein structures from sequence data banks.     

Structural characterization of alpha-helices of implicitly solvated poly-alanine

The structural characteristics of alpha-helices in poly-alanine-based peptides have been investigated via molecular dynamics simulation with the goal of understanding the basic features of peptide simulations within the context of a model system, classical molecular dynamics with generalized Born (GB) solvation, and to shed insight into the formation and stabilization of alpha-helices in short peptides. The effects of peptide length, terminal charges, proline substitution, and temperature on the alpha-helical secondary structure have been studied. The simulations have shown that distinct secondary structure begins to develop in peptides with lengths approaching 10 residues while ambiguous structures occur in shorter peptides. The helical content of peptides with lengths > or =10 amino acids is observed to be nearly constant up to (Ala)(40). Interestingly, terminal charges and proline in the second position from the N-terminus alter the secondary structure locally with little effect on the overall alpha-helical content of the peptide. The free energy profile of helix formation was also investigated. A large increase in free energy accompanying the formation of helices with more than two consecutive hydrogen bonds in the (i, i + 4) pattern was observed while the free energy increases linearly with additional hydrogen bonds. Values for the change in enthalpy and entropy of helix nucleation and propagation are reported. Additionally the results obtained from the GB model are compared to explicit solvent simulations of two synthetic alanine-based peptides.     

Conformational analysis of XA and AX dipeptides in water by electronic circular dichroism and 1H NMR spectroscopy

We measured the temperature-dependent electronic circular dichroism (ECD) spectra of AX, XA, and XG dipeptides in D2O. The spectra of all XA and AX peptides indicate a substantial population of the polyproline II (PPII) conformation, while the ECD spectra of LG, KG, PG, and AG were found to be quantitatively different from the alanine-based dipeptides. Additional UV absorption data indicate that the ECD spectra of the XG peptides stem from electronic coupling between the peptide and the C-terminal group, and that spectral differences reflect different orientations of the latter. We also measured the 1H NMR spectra of the investigated dipeptides to determine the 3JHalphaNH coupling constants for the C-terminal residue. The observed temperature dependence of the ECD spectra and the respective room-temperature 3JHalphaNH coupling constants were analyzed by a two-state model encompassing PPII and a beta-like conformation. The PPII propensity of alanine in the XA series is only slightly modulated by the N-terminal side chain, and is larger than 50%. As compared to AA, XA peptides containing L, P, S, K V, E, T, and I all cause a relative stabilization of the extended beta-strand conformation. The PPII fractions of XA peptides varied between 0.64 for AA and 0.58 for DA, whereas the PPII fractions of AX peptides were much lower. From the investigated AX peptides, only AL and AQ showed the expected PPII propensity. We found that AT, AI, and AV clearly prefer an extended beta-strand conformation. A quantitative comparison of AA, AAA, and AAAA revealed a hierarchy AAAA > AAA approximately AA for the PPII population, in agreement with predictions from MD calculations and results from Raman optical activity studies (McColl et al. J. Am. Chem. Soc. 2004, 126, 5076).     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

The conformation of tetraalanine in water determined by polarized Raman, FT-IR, and VCD spectroscopy

The present article reports the conformation of cationic tetraalanine in aqueous solution. The determination of the dihedral angles of the two central amino acid residues was achieved by analyzing the amide I' band profile in the respective polarized visible Raman, Fourier transform-IR, and vibrational circular dichroism (VCD) spectra by means of a novel algorithm which utilizes the excitonic coupling between the amide I modes of nearest neighbor and second nearest peptide groups. It is an extension of a recently developed theory (Schweitzer-Stenner, R. Biophys. J., 2002, 83, 523-532). UV electronic circular dichroism (ECD) spectra of the peptides were used to validate the results of the structure analysis. The analyses yielded the dihedral angles (phi(12), psi(12)) = (-70 degrees, 155 degrees ) and (phi(23), psi(23)) = (-80 degrees, 145 degrees ). The obtained values are very close to the Ramachandran coordinates of the polyproline II helix (PPII). The data suggest that this is the conformation predominantly adopted by the peptide at room temperature. This notion was corroborated by the corresponding electronic circular dichroism spectrum. Tetraalanine exhibits a higher propensity for PPII than trialanine for which a 50:50 mixture of polyproline II and an extended beta-strand-like conformation was obtained from recent spectroscopic studies (Eker et al., J. Am. Chem. Soc. 2002, 124, 14330-14341). The temperature dependence of the CD spectra rule out that any cooperativity is involved in the strand if PPII transition. This led to the conclusion that solvent-peptide interactions give rise to the observed PPII stability. Our result can be utilized to understand why the denaturation of helix-forming peptides generally yields a PPII rather than a heterogeneous random conformation.     

A 10-A spectroscopic ruler applied to short polyprolines

Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1-6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short F?rster radius (R0 ca. 9 A), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n > or = 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2-72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor-acceptor distances between ca. 8 A (n = 1) and ca. 16 A (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30-70 A in water and 220 +/- 40 A in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor-acceptor distances extracted by FRET are correlated with the F?rster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in F?rster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation.     

High-field 1H MAS and 15N CP-MAS NMR studies of alanine tripeptides and oligomers: distinction of antiparallel and parallel beta-sheet structures and two crystallographically independent molecules

beta-Strand peptides are known to assemble into either antiparallel (AP) or parallel (P) beta-sheet forms which are very important motifs for protein folding and fibril formations occurring in silk fibroin or amyloid proteins. Well-resolved 1H NMR signals including NH protons were observed for alanine tripeptides (Ala)3 with the AP and P structures as well as (Ala)n (n = 4-6) by high-field/fast magic-angle spinning NMR. Amide NH and amino NH3+ 1H signals of (Ala)3 with the P structure were well resonated at 7.5 and 8.9 ppm, respectively, whereas they were not resolved for the AP structure. Notably, NH 1H signals of (Ala)3 and (Ala)4 taking the P structure are resonated at higher field than those of the AP structure by 1.0 and 1.1 ppm, respectively. Further, NH 15N signals of (Ala)3 with the AP structure were resonated at lower field by 2 to 5 ppm than those of (Ala)3 with the P structure. These relative 1H and 15N hydrogen bond shifts of the P structure with respect to those of the AP structure are consistent with the relative hydrogen bond lengths of the interstrand N-H...O=C bonds. Distinction between the two crystallographically independent chains present in the AP and P structures was feasible by 15N chemical shifts but not by 1H chemical shifts because of insufficient spectral resolution in the latter. Calculated 1H and 15N shielding constants by density functional theory are generally consistent with the experimental data, although some discrepancies remain depending upon the models used.     

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.