Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.     

Neighbor effect on PPII conformation in alanine peptides

The polyproline II (PPII) conformation is dominant in short alanine oligomers. The noncooperativity of PPII structure in alanine peptides indicates that PPII in water is locally determined and that alanine neighbors are consistent with Flory's isolated pair hypothesis. However, neighbor effects from beta-branched or bulky aromatic residues tend to increase the Phi angle of the nearest neighbor as observed in coil library data. Here we demonstrate directly the neighbor effect using short alanine model peptides GGAAAGG, GGLnALnGG (Ln is norleucine), GGIAAGG, and GGIAIGG. The far-UV CD spectra, NMR 3JalphaN coupling constant, and H-D hydrogen exchange measurements reveal that Ile reduces the PPII content of the probe Ala side chain relative to Ala or norLeu. The free energy differences are consistent with predictions from electrostatic solvation free energy (ESF) calculations. The results indicate that prediction of PPII propensities or scales requires including the neighbor effect.     

The pentapeptide GGAGG has PII conformation

Most of what we know about proteins reflects their native folded structure. Much less is understood about the structure of unfolded proteins, which tends to be referred to as "random coil", lacking extended alpha-helix or beta-strand structure. Recent work suggests that unfolded proteins might adopt significant population of PII structure, an extended left-handed helix found in collagen and proline-rich peptides. A series of short peptides AcGGXGGNH2 has been adopted as a model for studying unfolded protein structure because of the minimal steric effect imposed by flanking glycines. Peptide AcGGAGGNH2 makes possible a host-guest conformation analysis of the middle residue alanine. NMR experiments reveal that the Phi and Psi dihedral angles of the central alanine are -73 degrees and 125 degrees , respectively, placing the alanine in the PII region of the Ramachandran plot. Circular dichroism shows a typical PII spectrum with a strong negative absorbance at 190 nm. Temperature experiments show the alanine structure shifts to increasing beta-strand at high temperature. Because the alanine side chain most closely represents unsubstituted peptide backbone, these results have significant implications for the conformational entropy of unfolded polypeptide chains.     

Solvent polarity controls the helical conformation of short peptides rich in Calpha-tetrasubstituted amino acids

The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.     

Conformation dependence of the CalphaDalpha stretch mode in peptides. 1. Isolated alanine peptide structures

Ab initio normal mode calculations have been performed on isolated alanine di- through octa-(i.e., blocked) peptides in uniform alphaR, beta, and polyproline II conformations to determine how the CalphaDalpha stretch mode, which has been proposed as a possible determinant of the varphi,psi conformation at the Calpha atom (Mirkin, N. G.; Krimm, S. J. Phys. Chem. A 2004, 108, 10923), depends on conformation and sequence length. This set of frequencies, including results on some kinked structures, demonstrates that such a discrimination is likely to be possible through experimental observations of peptides synthesized with successive deuteration at the Halpha sites, on the basis of at least three properties: the values of the frequency at the first residue, the pattern of successive frequency differences, and the frequency differences between the first and last residues.     

Factors affecting conformation in proline-containing peptides

[reaction: see text] NMR was used to study the thermodynamics of the cis --> trans isomerization for prolyl amide bonds in the compounds shown. The magnitude of K(t/c) for C-terminal esters is greater than for the corresponding amides, signifying stronger backbone stereoelectronic effects in esters. Increasing the steric bulk of the N-terminal residue from Ac- to Ac-Gly- favors the trans conformation. Incorporation of a Phe residue N-terminal to Pro, however, shifts the equilibrium in favor of the cis conformation, via a stabilizing aromatic-proline interaction.     

Conformational dependence of anharmonic vibrations in peptides: amide-I modes in model dipeptide

The conformational dependence of a set of anharmonic vibrational parameters for the amide-I modes in peptides has been examined at the level of Hartree-Fock theory. By using glycine dipeptide as a model molecule, the phi-psi maps of diagonal and off-diagonal anharmonicities, local mode mixing angle, zero-order local mode frequencies, as well as intermode coupling, were calculated, and all were found to exhibit certain conformational sensitivities. The characteristics of the phi-psi maps of the diagonal and off-diagonal anharmonicities were found to be complementary to each other, and the latter was found to correlate well with that of the mixing angle, reflecting the fact that these anharmonic parameters are interconnected and all determined by the same set of underlying anharmonic force field. The mean values of the diagonal and off-diagonal anharmonicities were found to be 15.7 cm(-1) and 9.9 cm(-1) respectively. The mean value for the two local mode frequency differences was estimated to be 9.7 cm(-1), showing a nondegenerate local mode picture. For significant peptide conformations, the calculated anharmonic parameters were found to be in reasonable agreement with values obtained at the level of density functional theory as well as values obtained with recent two-dimensional infrared experiments.     

Solvent dependence of electronic transition intensities

Most of the research on solvent effects on the electronic spectra of organic molecules has been concerned with frequency shifts. Solvent induced intensity changes, however, have not received much attention. At present there is no simple, general theory that relates: (a) the solvent induced changes in electronic transition intensities to (b) the macroscopic properties of the solvent. In this article we derive an equation that provides a quantitative interpretation for the changes in the absorption intensities. We show that solvent induced intensity changes are related to van der Waals constants for solvent and solute. Absorption data, some from previous investigations, are presented to show the usefulness and limitations of the theory.     

Solvent dependence of charge-transfer transitions in binary aqueous mixtures

Summary Charge-transfer transitions of Ru(bipy) ₃ ²⁺ , Fe(bipy) ₃ ²⁺ , Fe(CN) ₆ ^3- , and free bipy (bipy=2,2-bipyridine) are solvent dependent. Evidence is presented that dielectric continuum theory provides a reasonable basis for interpreting medium effects on the electronic transition energies in binary solvent mixtures as well as in pure solvents.     

Temperature dependence of the conformation of isotactic polystyrene in toluene

In order to obtain information about a possible helix–coil transition of isotactic polystyrene (i-PS) at 80°C in toluene, as has been reported in other solvents, solution properties were examined at temperatures between 10 and 110°C. Use was made of viscometry, high-resolution nuclear magnetic resonance, infrared spectroscopy, calorimetry, and light scattering. No distinct transition was found at 80°C but rather a second-order transition between 62 and 65°C. A similar transition occurred in toluene solutions of atactic polystyrene. The transition may be attributed to a sudden change in the mobility of the phenyl side-group of the polymer. From this study it is concluded that i-PS has a helical conformation in toluene, the mean helix length decreasing smoothly with increasing temperature.     

FTIR studies of collagen model peptides: complementary experimental and simulation approaches to conformation and unfolding

X-ray crystallography of collagen model peptides has provided high-resolution structures of the basic triple-helical conformation and its water-mediated hydration network. Vibrational spectroscopy provides a useful bridge for transferring the structural information from X-ray diffraction to collagen in its native environment. The vibrational mode most useful for this purpose is the amide I mode (mostly peptide bond C=O stretch) near 1650 cm-1. The current study refines and extends the range of utility of a novel simulation method that accurately predicts the infrared (IR) amide I spectral contour from the three-dimensional structure of a protein or peptide. The approach is demonstrated through accurate simulation of the experimental amide I contour in solution for both a standard triple helix, (Pro-Pro-Gly)10, and a second peptide with a Gly --> Ala substitution in the middle of the chain that models the effect of a mutation in the native collagen sequence. Monitoring the major amide I peak as a function of temperature gives sharp thermal transitions for both peptides, similar to those obtained by circular dichroism spectroscopy, and the Fourier transform infrared (FTIR) spectra of the unfolded states were compared with polyproline II. The simulation studies were extended to model early stages of thermal denaturation of (Pro-Pro-Gly)10. Dihedral angle changes suggested by molecular dynamics simulations were made in a stepwise fashion to generate peptide unwinding from each end, which emulates the effect of increasing temperature. Simulated bands from these new structures were then compared to the experimental bands obtained as temperature was increased. The similarity between the simulated and experimental IR spectra lends credence to the simulation method and paves the way for a variety of applications.     

Solvent viscosity dependence of the protein folding dynamics

Solvent viscosity has been frequently adopted as an adjustable parameter in various computational studies (e.g., protein folding simulations) with implicit solvent models. A common approach is to use low viscosities to expedite simulations. While using viscosities lower than that of aqueous is unphysical, such treatment is based on observations that the viscosity affects the kinetics (rates) in a well-defined manner as described by Kramers' theory. Here, we investigate the effect of viscosity on the detailed dynamics (mechanism) of protein folding. On the basis of a simple mathematical model, we first show that viscosity may indeed affect the dynamics in a complex way. By applying the model to the folding of a small protein, we demonstrate that the detailed dynamics is affected rather pronouncedly especially at unphysically low viscosities, cautioning against using such viscosities. In this regard, our model may also serve as a diagnostic tool for validating low-viscosity simulations. It is also suggested that the viscosity dependence can be further exploited to gain information about the protein folding mechanism.     

Solvent dependence of the spin-allowed transitions in free base tetra-t-butylphthalocyanine

The electronic absorption spectrum of tetra-t-butylphthalocyanine (TTBPc) was studied in various solvents. All bands were slightly red-shifted on increasing polarity of the solvents, indicating π—π* transitions. The oscillator strength of the Q_x band varied between 0.11 (t-butanol) and 0.27 (n-octane) and that of the Q_y band between 0.17 (t-butanol) and 0.28 (pyridine). The influence of the refractive index was masked by other solvent effects. Good agreement of the measured oscillator strength with modern theoretical values was obtained, when the random orientation of the solute molecules was taken into account.     

Cyclic peptides of sarcosine. Syntheses and conformation.

A series of cyclic peptides of sarcosine with the general formula c-Sar-n, n=2-8, has been synthesized and conformational studies carried out both in solution and in the solid. The rings are conformationally very homogeneous and contain both cis and trans amide bonds. Their barriers to ring inversion are high; in the smaller rings this is attributed to steric hindrance, caused by the N-methyl-groups, whilst in the larger rings the folding of the chain in helical segments plays an important role.     

Solvent dependence of intramolecular electron transfer in a helical oligoproline assembly

The helical oligoproline assembly CH3-CO-Pro-Pro-Pro-Pra(Ptzpn)-Pro-Pro-Pra(RuIIb2m2+ -Pro-Pro-Pra(Anq)-Pro-Pro-Pro-NH2, having a spatially ordered array of functional sites protruding from the proline backbone, has been prepared. The 13-residue assembly formed a linear array containing a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor with the proline II secondary structure as shown by circular dichroism measurements. Following RuII --> b2m metal-to-ligand charge-transfer (MLCT) excitation at 457 nm, electron-transfer quenching occurs, ultimately to give a redox-separated (RS) state containing a phenothiazine (PTZ) radical cation at the Pra(Ptzpn) site and an anthraquinone (ANQ) radical anion at the Pra(Anq) site. The redox-separated state was formed with 33-96% efficiency depending on the solvent, and the transient stored energy varied from -1.46 to -1.71 eV at 22 +/- 2 degrees C. The dominant quenching mechanism is PTZ reductive quenching of the initial RuIII(b2m*-) MLCT excited state which is followed by m*- --> ANQ electron transfer to give the RS state. Back electron transfer is highly exergonic and occurs in the inverted region. The rate constant for back electron transfer is solvent dependent and varies from 5.2 x 10(6) to 7.7 x 10(6) s-1 at 22 +/- 2 degrees C. It is concluded that back electron transfer occurs by direct ANQ*- --> PTZ*+ electron transfer. Based on independently evaluated kinetic parameters, the electron-transfer matrix element is HDA approximately 0.13 cm-1.