Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.     

Spectroscopic studies on the conformational stability of subtilisins in the presence of urea

Conformational stabilities of four alkaline bacterial proteases in the presence of 8 M urea at neutral pH were studied. The transitions were investigated by examining protein fluorescence, circular dichroism properties and catalytic activity. On the basis of their stability in urea solutions the enzymes could be ordered as follows: subtilisins Carlsberg and DY > subtilisin Novo > mesentericopeptidase.     

Electronic interactions and their influence on the conformational stability of trans-2-halocyclopentanol

Conformational preferences and electronic interactions of trans-2-fluorocyclopentanol (1), trans-2-chlorocyclopentanol (2), and trans-2-bromocyclopentanol (3) were analyzed using experimental and theoretical (3)J(HH) coupling constants, theoretical calculations, and natural bond orbital (NBO) analysis. The conformational equilibria of compounds 1-3 can be represented by their diaxial and diequatorial conformers as supported by theoretical calculations. From (3)J(HH) coupling constant values, it can be found that the diequatorial conformer is present in the equilibrium as 55% for compound 1 and as 60% for compounds 2 and 3. This behavior is in agreement with orbital interaction analyses obtained from NBO.     

Stereoelectronic effects on collagen stability: the dichotomy of 4-fluoroproline diastereomers

Collagen is the most abundant protein in animals. Natural collagen consists of a triple helix of (Xaa-Yaa-Gly)n chains, in which the Xaa and Yaa residues are often l-proline. Here, a (2S,4S)-4-fluoroproline (flp) residue is shown to be greatly stabilizing in the Xaa position (but destabilizing in the Yaa position). In contrast, a (2S,4R)-4-fluoroproline (Flp) residue is shown to be greatly destabilizing in the Xaa position (but stabilizing in the Yaa position). The dichotomous effect of the diastereomers appears to arise from a gauche effect, which alters pyrrolidine ring pucker and hence properly (or improperly) preorganizes main-chain dihedral angles. Thus, the rational use of stereoelectronic effects can enhance the conformational stability of a protein.     

Role of solvents in stability of collagen

The influence of hydroxymethyl chain length of the solvents on collagen was established with conformational stability and thermal stability. Thermal stability of monomeric collagen and RTT fibres (rat tail tendon) treated with methanol, ethylene glycol (EG) and glycerol were reported using the melting temperature for helix-coil transition and the peak temperature for collagen-gelatin transition. Both melting temperature and peak temperature increases as the hydroxymethyl chain length increases. Conformational stability of collagen solution treated with lower and higher concentrations of methanol, ethylene glycol and glycerol indicates that aggregation of collagen molecule is more at higher concentrations of these solvents. The concentration dependence is greater for the increased number of OH groups. Since protein aggregation is associated with neuro degenerative diseases, aggregation of collagen molecule in the presence of solvents is of great importance for biomedical application.     

On the effect of low concentrations of alcohols on the conformational stability of globular proteins

Low concentrations of alcohols have proven to be able to enlarge the stability curve of globular proteins, by decreasing the cold denaturation temperature and increasing the hot denaturation temperature [S. R. Martin, V. Esposito, P. De Los Rios, A. Pastore and P. A. Temussi, J. Am. Chem. Soc., 2008, 130, 9963-9970]. In order to try to explain these data, I have considered that: (1) an aqueous 2 M MeOH solution can be treated as a uniform liquid, constituted by water molecules, whose density, above the temperature of maximum density, has the same values of neat water, simply shifted by 2 °C toward lower temperatures, whereas, below the temperature of maximum density, it decreases to a slightly lesser extent than the density of neat water; (2) the ΔE(a)(2 M MeOH) quantity, a balance between intra-protein energetic attractions and those with the surrounding solvent molecules, both water and methanol, assumes a constant positive value. These physically-based assumptions, when inserted into the theoretical model developed to rationalize the occurrence of cold denaturation in neat water [G. Graziano, Phys. Chem. Chem. Phys., 2010, 12, 14245-14252], reproduce in a qualitatively correct manner the effect of low concentrations of alcohols.     

Nonempirical quantum-chemical study of the effect of substituents on the conformational stability of 3-substituted diaziridines

A full optimization of the geometry of 3-monosubstituted diaziridines with a range of substituents was made within the scope of the nonempirical form of the Hartree-Fock method using the 3-21G basis set. It was shown that the main factors affecting the destabilization of the 1,2-cis conformers are the interaction of the n_N orbitals and the steric repulsion of the hydrogen atoms.Scientific-Production Association, Institute of Applied Chemistry, Leningrad.Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 27, No. 4, pp. 459–462, July–August, 1991. Original article submitted October 12, 1990.     

The effect of sequence on the conformational stability of a model heteropolymer in explicit water

We investigate the properties of a two-dimensional lattice heteropolymer model for a protein in which water is explicitly represented. The model protein distinguishes between hydrophobic and polar monomers through the effect of the hydrophobic monomers on the entropy and enthalpy of the hydrogen bonding of solvation shell water molecules. As experimentally observed, model heteropolymer sequences fold into stable native states characterized by a hydrophobic core to avoid unfavorable interactions with the solvent. These native states undergo cold, pressure, and thermal denaturation into distinct configurations for each type of unfolding transition. However, the heteropolymer sequence is an important element, since not all sequences will fold into stable native states at positive pressures. Simulation of a large collection of sequences indicates that these fall into two general groups, those exhibiting highly stable native structures and those that do not. Statistical analysis of important patterns in sequences shows a strong tendency for observing long blocks of hydrophobic or polar monomers in the most stable sequences. Statistical analysis also shows that alternation of hydrophobic and polar monomers appears infrequently among the most stable sequences. These observations are not absolute design rules and, in practice, these are not sufficient to rationally design very stable heteropolymers. We also study the effect of mutations on improving the stability of the model proteins, and demonstrate that it is possible to obtain a very stable heteropolymer from directed evolution of an initially unstable heteropolymer.     

Thermal stability of collagen in intervertebral disc tissues

The thermal stability of collagen in intervertebral disc tissues was studied using differential scanning calorimetry. It was found that the melting of collagen in a native tissue was complete at 62–75°C (ΔH = 62.4 J/g) under heating excised annulus fibrosus and nucleus pulposus samples. On heating an intact structure up to 80°C, the denaturation of collagen did not occur. It was shown that the degradation of a proteoglycan component in the test tissues had no effect on the thermal stability of collagen.     

Thermodynamic conformational analysis and structural stability of the nicotinic analgesic ABT-594

This work presents a theoretical study of the nicotinic analgesic ABT-594. We describe its neutral (precursor) and protonated (active) forms in vacuum and aqueous solution at the MP2/cc-pVDZ level. A conformational analysis is performed on the two torsional angles describing the orientation of the azetidinyl group and the azetidinylmethoxy moiety. To account for entropic effects, a thermostatistical study of conformational populations at physiological temperature is carried out. In the neutral form, conformer I is found as the most populated in vacuum and solution. Here, the nitrogen of the azetidinyl group is far from the electron pairs of the oxygen and the pyridinic nitrogen. In the protonated form, conformer VIII is the most stable in vacuum and solution. Now, the additional proton on the azetidinyl group is oriented toward the electron lone pairs of oxygen. The structural stability of conformers I and VIII is considered through the atoms in molecules theory. The conformer I, in the neutral forms, is stabilized by an intramolecular hydrogen bond. The preference of conformer VIII in the protonated forms is explained by the higher strength of its intramolecular hydrogen bond over the cation-pi interaction found in conformer I. The effect of the interaction energy with the receptor on the conformational preferences of protonated ABT-594 is simulated. The result is that the population of conformers associated to the rotation of the azetidinyl group increases. So, the molecule can easily adopt the optimal internitrogen separation for interaction with the receptor.     

Investigation of conformational stability and vibrational spectra of halomethylsulfonyl isocyanates

The conformational behavior and structural stability of chloro- and fluoromethylsulfonyl isocyanates were investigated by quantum mechanical DFT and ab initio MP2 calculations. The 6-311++G^** basis set was employed to include polarization and diffuse functions in the calculations. The molecules were found to exist in a mixture of two stable gauche conformations. The potential scans were calculated from which the rotational barriers could be estimated. The vibrational frequencies and spectra were computed at B3LYP/6-311++G^** level. The potential energy distributions were then calculated to provide tentative vibrational assignment for the normal modes of the stable conformers of both molecules.     

Entropic control of the relative stability of triple-helical collagen peptide models

Herein, we show that current methodologies in atomistic simulations can yield reliable standard free energy values in aqueous solution for the transition from the dissociated monomeric form to the triple-helix state of collagen model peptides. The calculations are performed on a prototypical highly stable triple-helical peptide, [(Pro-Hyp-Gly)10]3 (POG10), and on the so-called T3-785 triple-helix mimicking a fragment from the type III human collagen, which is more thermally labile. On the basis of extensive MD simulations in explicit solvent followed by molecular-mechanical and electrostatic Poisson-Boltzmann calculations complemented with an accurate estimation of the nonpolar contributions to solvation, the computed free energy change for the aggregation processes of the POG10 and T3-785 peptides leading to their triple-helices is -6.6 and -6.1 kcal/mol, respectively. For POG10, this value is in agreement with differential scanning calorimetric data. However, it is shown that conformational entropy, which is estimated by means of an expansion of mutual information functions, preferentially destabilizes the triple-helical state of T3-785 by around 4.6 kcal/mol, thus explaining its lower thermal stability. Altogether, our computational results allow us to ascertain, for the first time, the actual thermodynamic forces controlling the absolute and relative stability of collagen model peptides.     

On the estimation of stability parameters from heat-induced conformational transition curves of proteins

A method is suggested to determine valid and authentic values of thermodynamic stability parameters of proteins from their heat-induced conformational transition curves. We show (a) that the estimate of ΔH_m ^van, the enthalpy change on denaturation at T_m, the midpoint of denaturation, is significantly less than ΔH_m ^cal, the value obtained by the calorimetric measurements, if the analysis of the conformational transition curve uses the conventional method which assumes a linear temperature-dependence of the pre- and post-transition baselines; and (b) that there exists an excellent agreement between ΔH_m ^van and ΔH_m ^cal values of proteins, if the analysis of thermal denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function. The latter analysis is supported by our observations that the temperaturedependencies of the absorption and circular dichroism properties of protein groups are indeed nonlinear. It is observed that the estimate of ΔC_p, the constant-pressure heat capacity change is independent of the model used to describe the temperaturedependence of the pre- and post-transition baselines. An important conclusion is that for proteins which exhibit a two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.     

Effects of conformational stability and geometry of guanidinium display on cell entry by beta-peptides

We have used our ability to control beta-peptide secondary structure in order to explore the effects of conformational stability and geometry of guanidinium display on cell entry. Both of these factors affect the rate and relative amount of beta-peptide accumulation in the cytoplasm and nucleus of live HeLa cells. These beta-peptides do not show significant differences in cell surface binding, implying that structure and guanidinium display are important in a later step in cell entry than initial surface binding.     

NMR monitoring of chain-specific stability in heterotrimeric collagen peptides

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15N/13C. Circular dichroism spectroscopy and differential scanning calorimetry thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. Heteronuclear single quantum coherence experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10 degrees C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.     

Insights into the conformational switching mechanism of the human vascular endothelial growth factor receptor type 2 kinase domain

Human vascular endothelial growth factor receptor type 2 (h-VEFGR2) is a receptor tyrosine kinase involved in the angiogenesis process and regarded as an interesting target for the design of anticancer drugs. Its activation/inactivation mechanism is related to conformational changes in its cytoplasmatic kinase domain, involving first among all the αC-helix in N-lobe and the A-loop in C-lobe. Affinity of inhibitors for the active or inactive kinase form could dictate the open or closed conformation of the A-loop, thus making the different conformations of the kinase domain receptor (KDR) domain different drug targets in drug discovery. In this view, a detailed knowledge of the conformational landscape of KDR domain is of central relevance to rationalize the efficiency and selectivity of kinase inhibitors. Here, molecular dynamics simulations were used to gain insight into the conformational switching activity of the KDR domain and to identify intermediate conformations between the two limiting active and inactive conformations. Specific energy barriers have been selectively removed to induce, and hence highlight at the atomistic level, the regulation mechanism of the A-loop opening. The proposed strategy allowed to repeatedly observe the escape of the KDR domain from the DFG-out free energy basin and to identify rare intermediate conformations between the DFG-out and the DFG-in structures to be employed in a structure-based drug discovery process.     

Density functional theory investigations on the conformational stability and vibrational spectra of the model compound of vitamin K

Becke 3-Lee–Yang–Parr density functional theory (DFT/B3LYP) using 6-31G(d) and 6-311G(d) basis sets and the scaled quantum mechanics (SQM) force field method are used to study molecular conformations and vibrational spectra of a model compound of vitamin K (VK). In this molecule, there are six conformers on the torsional potential energy map of the dihedral angles C₈C₁₄C₁₅C₁₆ () and C₇C₈C₁₄C₁₅ (β). It is shown that the VK_1 conformer ( = 237.7° and β = 274.2°) is the most stable form. For this lowest energy conformer, the harmonic force fields calculated by B3LYP/6-311G(d) and B3LYP/6-31G(d) methods are scaled with one scale factor of 0.9623 and a set of different scale factors transferred from the previous studies for the other similar molecules, respectively. The vibrational frequencies, IR intensities, and Raman activities are obtained for the lowest energy conformer. On the basis of B3LYP calculations, the normal mode analysis is performed to assign the vibrational fundamental frequencies according to the potential energy distributions. The computational frequencies are in good agreement with the observed results.