Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine‐rich DNA sequences that may form G‐quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G‐quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G‐quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G‐rich sequences in the presence or the absence of their complementary C‐rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26‐meric human telomeric sequence d[A₃G₃(T₂AG₃)₃A₂]. In the presence of K⁺ ions, the latter adopts the hybrid‐1 G‐quadruplex conformation, a tightly packed structure with an unusually small number of solvent‐exposed atomic groups. The K⁺‐induced folding of the G‐quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G‐quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G‐quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G‐quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014.     

Probing hydration of monovalent cations condensed around polymeric nucleic acids

We report the relative molar sound velocity increments, [U], partial molar volumes, V(o), and partial molar adiabatic compressibilities, K(S)(o), of the Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and N(CH(3))(4)(+) salts of poly(dAdT)poly(dAdT), poly(dGdC)poly(dGdC), poly(dIdC)poly(dIdC), poly(rA)poly(rU), poly(rG)poly(rC), poly(rI)poly(rC), and poly(rU) at 25 degrees C. When analyzing these data, we take into account the Donnan membrane equilibrium effect. Comparison between the values of [U], V(o), and K(S)(o) exhibited by the nucleic acid salts and respective chlorides (LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and N(CH(3))(4)Cl) yields information about the state of counterion hydration in the vicinity of each nucleic acid structure studied here. Our analysis reveals that the poly(dGdC)poly(dGdC), poly(dIdC)poly(dIdC), and poly(rI)poly(rC) duplexes and single-stranded poly(rU) do not significantly influence the hydration properties of their condensed counterions. In the vicinity of these polymers, counterions retain their full hydration shells (within +/-15%). By contrast, counterions condensed around the poly(dAdT)poly(dAdT), poly(rA)poly(rU), and poly(rG)poly(rC) duplexes are significantly dehydrated and retain, respectively, only 65(+/-18)%, 34(+/-21)%, and 33(+/-9)% of their original hydration shells. Taken together, the volumetric data reported here provide important new information that ultimately may help us understand the central role that hydration and counterions play in modulating the conformational preferences of nucleic acids and the energetics of DNA recognition events.     

Interactions between homopolymeric amino acids (HPAAs)

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.     

Comparative analysis of the cytotoxicity of homopolymeric amino acids

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.     

Compressibility changes accompanying conformational transitions of apomyoglobin

We used high-precision density and ultrasonic velocity measurements to characterize the native (N), molten globule (MG), and unfolded (U) conformations of apomyoglobin. The molten globule states that were studied in this work include the MG(pH4)(NaCl) state observed at pH 4 and 20 mM NaCl, the MG(pH4)(NaTCA) state observed at pH 4 and 20 mM sodium trichloracetate (NaTCA), the MG(pH2)(NaCl) state observed at pH 2 and 200 mM NaCl, and the MG(pH2)(NaTCA) state observed at pH 2 and 20 mM NaTCA. We used our densimetric and acoustic data to evaluate changes in adiabatic compressibility associated with the acid- or salt-induced N-to-MG, MG-to-U, MG-to-MG, and U-to-MG transitions of the protein. The N-to-MG(pH4)(NaCl) and N-to-MG(pH4)(NaTCA) transitions are accompanied by decreases in compressibility of -(3.0 +/- 0.6) x 10(-6) and -(2.0 +/- 0.6) x 10(-6) cm3 g(-1)bar(-1), respectively. The N-to-MG(pH2)(NaCl) and N-to-MG(pH2)(NaTCA) transitions are associated with compressibility changes of -(4.9 +/- 1.1) x 10(-6) and (0.7 +/- 0.9) x 10(-6) cm3 g(-1) bar(-1), respectively. We interpret these data in terms of the degree of unfolding of the various molten globule forms of apomyoglobin. In general, our compressibility data reveal significant disparities between the various equilibrium molten globule states of apomyoglobin while also quantitatively characterizing each of these states. Volumetric insights provided by our data facilitate gaining a better understanding of the folding pathways, intermediates, and kinetics of apomyoglobin folding.     

Compressibility,densimetric and calorimetric studies of hydration of carrageenans in the random form

The partial molal volume and adiabatic compressibility were measured, as well as their counterion activity, for sodium and potassium salts of three types of carrageenan (κ-, ι- and λ-components) in aqueous solutions at 25°C. Furthermore, the amount of related unfreezable water was estimated by the differential scanning calorimetry. On the basis of these results, the hydration states of carrageenans in the random form were comparatively discussed in relation to their chemical structure, counterion binding and polymer concentration. The sodium salt of each component showed a larger amount of hydration when compared with the corresponding potassium salt. The amount of hydration estimated from molal volume and compressibility data (in dilute solution) increased in the order of κ < ι < λ, while the amount of unfreezable water (in concentrated solution) decreased in the same order. These characteristics hydration behaviours of carrageenans seemed to be reasonably explained in terms of the effects of the charge density and counterion dissociation of these polyions.     

Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.     

The origin of chirality in protein amino acids

We discuss the origin of the chirality of protein amino acids from the point of view of a phase transition from a racemic mixture into an optically pure state. We assume that Bose–Einstein condensation may act as an amplification mechanism. The original theory is due to Salam. We suggest a new role for the phase transition. Following Quack we distinguish parity violation of two kinds (de facto and de lege symmetry breaking). While the Salam phase transition corresponds to parity violation of the second kind (de lege), the phase transition we discuss in this work corresponds to parity violation of what we may call a third kind. This is suggested by recent experimental phenomena which correlate chiral symmetry breaking and pattern formation (spontaneous symmetry breaking that separates an initial racemic mixture into right- and left-handed space domains by means of a substrate). Tentative comments are given on the eventual design of possible experiments that may test this new hypothesis. © 1994 Wiley-Liss, Inc.     

Polycondensation of thermal precursors of amino acids and characterization of constituent amino acids.

As a model reaction of polyamino acid formation from non-amino acid precursors, diammonium citraconate (I), ammonium citraconamate(II) and ammonium itaconamate(III) were converted to polyimide type polymers by thermal polycondensation by heating at 130–210°C. The imide type polymer was partially hydrolyzed to the corresponding peptide type polyamino acid. The polymer was composed of α-methylaspartic acid (IV), threo- and erythro-ß-methylaspartic acid (V) and α-(aminomethyl) succinic acid (VI). On the other hand, IV was thermally polycondensed to the corresponding polymer. It was found that the amino acid composition of the polymer was similar to that of the polymer prepared from I, II and III. The formation and isomerization of amino acids during the thermal polycondensation are described.     

Volumetric effects of ionization of amino and carboxyl termini of alpha,omega-aminocarboxylic acids

We have determined the partial molar volumes and adiabatic compressibilities of a homologous series of six alpha,omega-aminocarboxylic acids over a broad pH range at 25 degrees C. We interpret the resulting data in terms of the changes in hydration associated with neutralization of amino and carboxyl termini. By combining our volumetric results with pH-dependent data on 1-anilinonaphthalene-8-sulfonic acid fluorescence we propose the following explanation to the long-standing observation that changes in volume and compressibility accompanying neutralization of a carboxyl group depend on the type of the solute in contrast to solute-independent changes in these parameters accompanying neutralization of an amino group. Unlike amino groups, neutralized carboxyl groups are capable of forming hydrogen-bonded structures stabilized by hydrogen bonds between the carbonyl oxygen of one solute molecule and the hydroxyl group of another molecule. Formation of such hydrogen-bonded structures causes an additional decrease in solute hydration with concomitant increases in volume and compressibility. Furthermore, solutes with large aliphatic moieties may form larger associates stabilized, in addition to intermolecular hydrogen bonds, by hydrophobic interactions which will result in further increases in volume and compressibility. In the aggregate, our results emphasize the need for further studies focused on developing an understanding of the role of electrostatic interactions in stabilizing/destabilizing proteins and protein complexes.     

The Genetic Algorithm and the Conformational Search of Polypeptides and Proteins

Abstract

The genetic algorithm is a technique of function optimization derived from the principles of evolutionary theory. We have adapted it to perform conformational search on polypeptides and proteins. The algorithm was first tested on several small polypeptides and the 46 amino acid protein crambin under the AMBER potential energy function. The probable global minimum conformations of the polypeptides were located 90% of the time and a non-native conformation of crambin was located that was 150kcal/mol lower in potential energy than the minimized crystal structure conformation. Next, we used a knowledge-based potential function to predict the structures of melittin, pancreatic polypeptide, and crambin. A 2.31 Å ΔRMS conformation of melittin and a 5.33 Å ΔRMS conformation of pancreatic polypeptide were located by genetic algorithm-based conformational search under the knowledge-based potential function. Although the ΔRMS of pancreatic polypeptide was somewhat high, most of the secondary structure was correct. The secondary structure of crambin was predicted correctly, but the potential failed to promote packing interactions. Finally, we tested the packing aspects of our potential function by attempting to predict the tertiary structure of cytochrome b ₅₆₂ given correct secondary structure as a constraint. The final predicted conformation of cytochrome b ₅₆₂ was an almost completely extended continuous helix which indicated that the knowledge-based potential was useless for tertiary structure prediction. This work serves as a warning against testing potential functions designed for tertiary structure prediction on small proteins.     

Aqueous solutions containing amino acids and peptides. Part 20. Volumetric behavior of some terminally substituted amino acids and peptides at 298.15 K

The densities at 298.15 K of aqueous solutions containing some terminally substituted amino acids and peptides containing the glycyl, L -and D -alanyl, L -leucyl, sarcosyl, and L -prolyl residues have been dertermined and standard state partial molar volumes and volumetric pairwise virial coefficients obtained. It is shown that the partial molar volumes can be represented using group volume contributions, but this approach is only approximate, and significant effects of N-terminal substitution and sequence dependence are observed. The volumetric virial coefficients for the amino acid amides have been expressed using a group-additivity approach, and the results obtained indicate that the dominant contributions come from peptide group interactions with other peptide groups and with hydrophobic groups. There is also some evidence of both sequence and chiral effects on the volumetric virial coefficients for proline-containing dipeptides.     

Synthesis and characterization of polyamides containing unnatural amino acids

We describe the synthesis of several polyamides that retain the secondary structure of proteins and contain derivatizable side chains. The derivatizable side chain allows for further reaction of the polymer chain (e.g., chain cross-linking or addition of pendant groups). Polymers of α-amino acids containing a terminal unsaturated bond on the side chain have been synthesized. Poly-L -pentenyl glycine, poly-L -propargyl glycine, and poly-L -allyl glycine were synthesized chemically via Leuchs' anhydrides and enzymatically using subtilisin Carlsberg. Poly-L -propargyl glycine and poly-D ,L -allyl glycine folded into the β-sheet configuration whereas poly-L -pentenyl glycine assumed a helical conformation. The secondary structure of poly-L -allyl glycine and poly-D ,L -pentenylglycine could not be determined conclusively. Comparison of properties between the polymers obtained chemically and enzymatically is provided. © 1995 John Wiley & Sons, Inc.     

Electronic and vibrational circular dichroism of aromatic amino acids by density functional theory

Structures of model compounds mimicking aromatic amino acid residues in proteins are optimized by density functional theory (DFT), assuming that the main-chain conformation was a random coil. Excitation energies and dipole and rotational strengths for the optimized structures were calculated based on time-dependent DFT (TD-DFT). The electronic circular dichroism (ECD) bands of the models were significantly affected by side-chain conformations. Hydration models of the aromatic residues were also subjected to TD-DFT calculations, and the ECD bands of these models were found to be highly perturbed by the hydration of the main-chain amide groups. In addition to calculating the random-coil conformation, we also performed TD-DFT calculations of the aromatic residue models, assuming that the main-chain conformation was an alpha-helix or beta-strand. As expected, the overall feature of the ECD bands was also perturbed by the main-chain conformations. Moreover, vibrational circular dichroism (VCD) spectra of the hydration models in a random-coil structure were simulated by DFT, which showed that the VCD spectra are more sensitive to the side-chain conformations than the ECD spectra. The present results show that analyses combining ECD and VCD spectroscopy and using DFT calculations can elucidate the main- and side-chain conformations of aromatic residues in proteins.     

Chain conformation in polyretropeptides III: Design of a 310 helix using α,α-dialkylated amino acids and retropeptide bonds

Computer simulations have been used to design a polypeptide with a 3₁₀ helix conformation. The study has been been performed taking advantage of the intrinsic helix forming tendency of α-Aminoisobutyric acid. In order to avoid the formation of the α helix, which is the other common helical conformation adopted by α-Aminoisobutyric acid-based peptides, retropeptide bonds have been included in the sequence. Thus, retropeptides are not able to form the intramolecular hydrogen bonding interactions characteristic of the α helix. The influences of both the peptide length and the solvent have been examined and compared with those of the polypeptide without retropeptide bonds. Proteins 29:575–582,1997. © 1997 Wiley-Liss, Inc.     

Infrared studies of water induced conformational changes in bacteriorhodopsin

Fourier transform infrared difference spectroscopy has been used to study the effect of water on the conformation of bacteriorhodopsin. The infrared spectra as a function of water content show a conformational change at about 0.06 g H₂O/g bacteriorhodopsin. By an interference method the thickness of the sample was measured and shows similar behavior as a function of water content. This study gives insight into the process of water absorption by purple membrane. The observations are in good agreement with those found for other proteins.Abbreviations IR infrared - FTIR Fourier transform IR     

Energetics of superhelicity and of B-Z transitions in superhelical DNA

The linking difference, α, imposed upon a superhelically constrained DNA molecule must be partitioned between twisting and bending deformations. Transitions to alternative secondary structures can occur at susceptible sites, altering the local molecular twist by an amount ΔTw _trans. That part of the linking difference not accommodated in this way, the residual linking difference α_res, must be manifested as smooth torsional and flexural deformations of secondary structure. The competition among the alternative ways of accommodating the imposed linking difference α determines a stressed equilibrium state. The superhelical free energy,G(α), is the excess free energy of the equilibrium state at linking difference α above that of the relaxed state under identical conditions. In this paper a method is described by which the free energies associated both to linking,G(α), and to residual linking differences can be determined from data on superhelical conformational transitions. The application of this approach to previously published experimental data on the B-Z transition suggests that the free energy associated with α_res is about 30% larger at substantial superhelicities than it is near the relaxed state. At the onset of transition the functional form ofG(α) is shown to change in a manner dependent upon the length of the Z-susceptible site.     

Pressure dependence of the apparent specific volume of bovine serum albumin: Insight into the difference between isothermal and adiabatic compressibilities

There are some theoretical arguments related to interpreting the adiabatic compressibility (β_s) of a protein determined from the sound velocity and the difference between β_s and isothermal compressibility (β_T). To address these problems experimentally, we constructed a high-pressure oscillating densitometer and used it to measure the apparent specific volume of bovine serum albumin as a function of pressure (0.1–78 MPa) and temperature (5–35 °C). The β_T determined from plots of the apparent specific volume vs. pressure was slightly larger than β_s at all temperatures examined, with the difference between the two compressibilities increasing as the temperature was decreased. Only at room temperature did the observed β_T agree with those estimated from β_s using the heat capacity and the thermal expansibility of the protein, suggesting that there are significant as-yet-unknown mechanisms that affect protein compressibility.     

Conformational stability of biological polyelectrolytes: Evaluation of enthalpy and entropy changes of conformational transitions

A new method is proposed for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution. For all transition midpoints, defined by given temperature and ionic strength, the total free energy change of the system is zero, which means that the nonionic contribution to the free energy change is equal in value and opposite in sign to the polyelectrolytic one. The counterion condensation theory of linear polyelectrolytes provides for the appropriate analytical expression to be used in such calculations. Linear plots of the proper functions of the calculated free energy changes vs the proper functions of temperature allows for the determination of the enthalpic and entropic terms of the nonionic free energy change of transition. The method has been applied to the extensive available data of the ion-induced conformational change of κ-carrageenan, a linear sulfated galactan extracted from seaweeds. The method has proved very successful, with the results showing a remarkable convergency of the enthalpy values for different monovalent counterions. On the other hand, the above approach has made it possible to explain the known effect of counterion specificity on the transition by a small difference in the nonionic entropic contributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 203–216, 1998