Distribution of charged residues stabilizes individual helices in myoglobins

The effect of the distribution of charged residues on stability of alpha helices in isolated peptides and in globular proteins exemplified by myoglobins from 62 different species is discussed. A highly simplified set of rules is used to account for the interaction of charged groups with the dipole of an alpha helix. Only the position and sign of a charge with respect to the center of the helix and its ability to participate in intrahelical salt bridges determine its effect. These rules lead to a linear correlation between the helicity in variant C-peptide helices from RNAse and the extent to which the charge distribution opposes the helix dipole. Of the sample of 496 helices in the myoglobins studied, 456 exhibit arrangements of charges which oppose the effective dipole moment of the helix according to this calculation. A number of variants occur which leave the backbone moment of helices A-D unchanged, or even add to it. However no such variants exist in the sequences of helices E-H. We suggest that the E, F, G and H helices in myoglobins which show the strongest reversal of the helix dipole participate in the structures of early intermediates in folding of the chain. Stable helix structures should be more likely to occur in these isolated sequences also, and introduction of charge alterations in helices E to H should affect the initial refolding rate of mutant myoglobins.     

The alpha helix dipole: screened out?

Aligned alpha helix peptide dipoles sum to a "macroscopic" dipole parallel to the helix axis that has been implicated in protein folding and function. However, in aqueous solution the dipole is counteracted by an electrostatic reaction field generated by the solvent, and the strength of the helix dipole may reduce drastically from its value in vacuum. Here, using atomic-detail helix models and Poisson-Boltzmann continuum electrostatics calculations, the net effective dipole moment, mu(eff), is calculated. Some initially surprising results are found. Whereas in vacuum mu(eff) increases with helix length, the opposite is found to be the case for transmembrane helices. In soluble proteins, mu(eff) is found to vary strongly with the orientation and position of the helix relative to the aqueous medium. A set of rules is established to estimate of the strength of mu(eff) from graphical inspection of protein structures.     

The determination of the thermodynamic parameters of the helix-coil transition of polypeptides by means of dipole moment measurements

The theoretical change of the mean-square dipole moment of a polypeptide during the helix-coil transition is compared with the change in helix content. It is shown that, according to the theory, the determination of the helix initiation parameter σ and the enthalpy of helix formation ΔH can be determined. Experimental data on poly-benzyl-L -gluatamate in two different solvent mixtures are given.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

Structure of aggregates and helix-coil transition of polypeptides by dielectric measurements. II. Poly-D,L-phenylalanine and poly-beta-benzyl-L-aspartate in chloroform-dichloroacetic acid mixtures

The dielectric absorption of poly-DL -phenylalanine and poly-γ-benzyl-L -aspartate (PLAB) was measured in very dilute solutions to determine the type of molecular association and to locate the helix–coil transition. Both polypeptides were present as associated helices in chloroform. The mode of aggregation, which was determined by measuring the dipole moment and the critical frequency, did not depend on the polarity of the side chain but rather on that of the solvent. In both polymers, the dissociation of the aggregates in chloroform was observed on addition of small amounts of dichloroacetic acid; further addition of the acid lead to the helix–coil transition. No second region of dielectric absorption that might be related to the kinetics of the transition was found during the helix–coil transition of PBLA.     

Helix-helix interactions in lipid bilayers.

Using a continuum model, we calculated the electrostatic interaction free energy between two alpha-helices in three environments: the aqueous phase, a low dielectric alkane phase, and a simple representation of a lipid bilayer. As was found in previous work, helix-helix interactions in the aqueous phase are quite weak, because of solvent screening, and slightly repulsive, because of desolvation effects that accompany helix assembly. In contrast, the interactions can be quite strong in a hypothetical alkane phase because desolvation effects are essentially nonexistent and because helix-helix interactions are not well screened. In this type of environment, the antiparallel helix orientation is strongly favored over the parallel orientation. In previous work we found that the free energy penalty associated with burying helix termini in a bilayer is quite high, which is why the termini tend to protrude into the solvent. Under these conditions the electrostatic interaction is strongly screened by solvent; indeed, it is sufficient for the termini to protrude a few angstroms from the two surfaces of the bilayer for their interaction to diminish almost completely. The effect is consistent with the classical model of the helix dipole in which the dipole moment is represented by point charges located at either terminus. Our results suggest, in agreement with previous models, that there is no significant nonspecific driving force for helix aggregation and, hence, that membrane protein folding must be driven by specific interactions such as close packing and salt-bridge and hydrogen bond formation.     

Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate.

The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.     

Ab initio molecular orbital study of the mispairing ability of a nucleotide base analogue, N4-aminocytosine

The intrinsic properties of N4-aminocytosine, a base analogue of cytosine, are analyzed by an ab initio molecular orbital method. Relative stabilities of four possible isomeric structures of N4-aminocytosine are shown. The more stable isomer has the smaller dipole moment, so the relative stabilities of the isomers in solutions are subject to solvent polarity. The mutagenicity of this base analogue must arise because it can behave like either cytosine or thymine. It can form a guanine-cytosine-like base pair more easily than cytosine, and an adenine-thymine-like base pair less easily than thymine.     

The structure and dipole moment of globular proteins in solution and crystalline states: use of NMR and X-ray databases for the numerical calculation of dipole moment

The large dipole moment of globular proteins has been well known because of the detailed studies using dielectric relaxation and electro-optical methods. The search for the origin of these dipolemoments, however, must be based on the detailed knowledge on protein structure with atomic resolutions. At present, we have two sources of information on the structure of protein molecules: (1) x-ray databases obtained in crystalline state; (2) NMR databases obtained in solution state. While x-ray databases consist of only one model, NMR databases, because of the fluctuation of the protein folding in solution, consist of a number of models, thus enabling the computation of dipole moment repeated for all these models. The aim of this work, using these databases, is the detailed investigation on the interdependence between the structure and dipole moment of protein molecules. The dipole moment of protein molecules has roughly two components: one dipole moment is due to surface charges and the other, core dipole moment, is due to polar groups such as N--H and C==O bonds. The computation of surface charge dipole moment consists of two steps: (A) calculation of the pK shifts of charged groups for electrostatic interactions and (B) calculation of the dipole moment using the pK corrected for electrostatic shifts. The dipole moments of several proteins were computed using both NMR and x-ray databases. The dipole moments of these two sets of calculations are, with a few exceptions, in good agreement with one another and also with measured dipole moments.     

Use of protein database for the computation of the dipole moments of normal and abnormal hemoglobins.

Previously, we discussed the calculation of the dipole moments of small proteins using the three-dimensional protein data-base. Our results demonstrate that the calculated dipole moments are in acceptable agreement with measured values. We, however, noted the difficulty of the calculation with larger proteins, in particular those consisting of several subunits. Hemoglobin (Hb) is a protein having a molecular weight of 64,000 that consists of four subunits, a typical case where the computation was found to be difficult. To circumvent the difficulties, we calculated the dipole moment of each subunit separately. The dipole moment of the whole protein was calculated by the vectorial summation of subunit moments. With this method, the calculated net dipole moment is in good agreement with the experimental value. Our calculation shows that the dipole moment vectors of subunits are, by and large, antiparallel in tetramers causing partial cancellation of the net dipole moment. In addition to normal HbA, the dipole moment of abnormal HbS was calculated using an approximate computational technique. Because of the loss of two negative changes as a result of the replacement of glutamic acid with valine in beta-chains, the dipole moment of HbS was found, experimentally and theoretically, to be significantly smaller than that of HbA.     

The dipole moments of membrane proteins: potassium channel proteins. II. T1 assembly. Search for the voltage sensor

The dipole moments of potassium channel protein (Kcsa) and beta-subunits were discussed in the previous paper of this series [Takashima, Biophys. Chem. 94 (2001) 209-218]. While the dipole moment of beta-subunits was found to be very large, the dipole moment of Kcsa turned out to be somewhat smaller than beta-subunits. As the continuation of this work, the discussion of the present paper is focussed on the dipole moment of T1 assembly, another component of the K-channel. As discussed later, the calculation using the X-ray crystallographic data by MacKinnon et al., revealed an astoundingly large dipole moment for T1 assembly. The dipole moment of T1 assembly combined with the likewise large dipole moment of beta-subunits amounts to a sufficient value to play an essential role as a voltage sensor of potassium channel.     

Position-dependent interactions between cysteine residues and the helix dipole

A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pK(a) between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family.     

Electrostatic potentials of the alpha helix dipole and of elastase

Molecular graphics has been used to display the electrostatic potentials of the α-helix dipole and that of elastase calculated using atomic charges obtained by a new, simple method^1–3. Calculations on the α-helix dipole support the simple dipole model in which the helix is represented by single, half integral charges at the helix termini. The potentials of elastase show some interesting features which may be related to the binding processes.     

Electric properties and structure of DNA-restriction fragments from measurements of the electric dichroism

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.     

Electrostatic interactions between α-helix dipoles in crystals of an uncharged helical undecapeptide

The electrostatic interactions between α-helix dipoles in the crystals of an uncharged helical undecapeptide have been studied in detail. The electrostatic interaction energy between one helix dipole and its 26 nearest neighbors is approximately ?23 kcal mol^?1. A very similar result is obtained when calculating the interactions between one helix dipole and all 988 helix dipoles occurring within a distance of 75 Å. It therefore appears that in these crystals of completely uncharged molecules large, favorable electrostatic interactions occur.     

Error propagation across levels of organization: from chemical stability of ribosomal RNA to developmental stability

My hypothesis integrates molecular and whole-organism levels of development. A physico-chemical property of nucleotides (their dipole moment), confers structural thermostability on double-stranded sequences, and decreases chemical stability of single-stranded sequences. According to this approach, low ribosomal RNA stability should decrease the precision of protein synthesis and whole-organism developmental stability. Indeed, substitution frequencies in pseudogenes are proportional to the subtraction of the dipole moment of the substituting nucleotide from that of the substituted one, and developmental instability, estimated by morphological fluctuating asymmetry (FA), correlates with mammal 12s rRNA base content of loop (but not stem) regions. In lizards, fit to the single-strand rationale of sequence chemical stability decreases with the level of poikilothermy of the investigated lizard family, suggesting interactions between changes in body temperature, ribosomal structure and developmental instability. Results confirm the hypothesis (less than for 12s rRNA) in: third codon positions of cytochrome B, probably because, unlike rRNAs, specific mRNAs affect only the protein they code; and 16s rRNA, apparently because its base composition is more affected by genome-wide mutational biases than that of 12s rRNA.     

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.     

Conformational flexibility of the furanose ring in DNA and its dipole moment

The influence of conformational rearrangement of the furanose ring in DNA on its dipole moment is studied. The dipole moment of the deoxyribose molecule as a function of its puckered state is calculated by the quantum-mechanical method using the MINDO/3 approximation. The values of the dipole moment and its components are obtained at various magnitudes of the pseudorotation phase angle. The C3'-endo in equilibrium C2'-endo conformational transition of deoxyribose is shown to be accompanied by the change in the dipole moment up to 3D. The results obtained are used to explain the structural properties of the DNA hydration shell.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.