Side chain contributions to the stability of alpha-helical structure in peptides.

Short peptides that contain significant alpha-helical structure in aqueous solution allow the investigation of the role of amino acid side chains in stabilizing or destabilizing alpha-helix structure. A host-guest system of soluble synthetic peptides was designed that consisted of chains with the block sequence TyrSerGlu4Lys4X3Glu4Lys4, denoted EXK, in which X represents any "guest" amino acid residue. Circular dichroism spectroscopy indicates that the extent of helicity of these peptides follows the order Ala greater than Leu greater than Met greater than Gln greater than Ile greater than Val greater than Ser greater than Thr greater than Asn greater than Gly. This order differs from both host-guest copolymer values (Met greater than Ile greater than Leu greater than Ala greater than Gln greater than Val greater than Thr greater than Asn greater than Ser greater than Gly) and the tendencies of these amino acids to occur in helices in globular proteins (Ala greater than Met greater than Leu greater than Gln greater than Ile greater than Val greater than Asn, Thr greater than Ser greater than Gly), but matches the order found in a series of synthetic coiled-coil alpha helices, except for Ser. Proton nuclear magnetic resonance analysis of several EXK peptides indicates that these peptides are partially helical, with the helical residues favoring the amino terminus.

DNA binding by single HMG box model proteins.

The HMG1/2 family is a large group of proteins that share a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg(2+). In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg(2+) is present in the nucleus and these proteins are expressed at a very high level.

Conformation and thermodynamics of DNA "necks". Models for three-arm branch formation in a duplex.

The properties of three-arm DNA junctions differ from those of four-arm junctions in several respects. Most apparently, bases flanking the branch are reactive to single strand specific agents in three-arm junctions but not four-arm junctions. To determine the basis for this, we have designed and synthesized a series of complexes in which a short duplex, a neck, progressively extends from a parent 16-mer DNA duplex. These structure allow us to investigate how a three-arm branch forms, and how its properties change as the neck extends. Comparison of the properties of a nicked duplex with those of the neck structures using native gel electrophoresis with reporter arms attached reveals progressively greater geometrical perturbation of the complexes as the number of base-pairs in the neck increases. Footprinting by single-strand specific reagents indicates that the reactivity to single-strand reagents near the branch occurs when only a single pair is possible. The branch in each neck interacts tightly with ethidium, as does a nick in the same duplex. The thermodynamics of neck formation have been evaluated by calorimetry and from the concentration dependence of absorbance temperature profiles. Each neck complex is destabilized with respect to duplex DNA or a nicked duplex, and has a lower enthalpy of formation despite the increased number of base-pairs present. A model is proposed to account for these properties in which the bases in the pair adjacent to the duplex interact directly with the duplex, via transient insertion.

Parallel and antiparallel Holliday junctions differ in structure and stability.

Two Holliday junction analogs, JA and JP, containing identical base-paired arms have been constructed from oligonucleotides. The former is constrained to adopt an antiparallel Sigal-Alberts structure, and the latter a parallel structure, by means of single strand d(T)9 tethers. We evaluate here the free energy difference between JA and JP using two different methods. One is a direct measurement of the ratio of the equilibrium constants for formation of branched structures from intact duplexes using one labeled strand and a competition assay. The second method estimates the difference in stability from the difference in thermal denaturation temperatures of JA and JP, using urea to shift the tm of the complexes. Both methods reveal a small free energy difference between the two complexes: JA is more stable than JP by -1.1(+/- 0.4) kcal (mol junction)-1, at 25 degrees C, 5 mM-Mg2+, from the first method, and by -1.6(+/- 0.3) kcal (mol junction)-1, according to the second. DNase I and the resolvase, endonuclease I from phage T7, cleave JA differently from JP in the vicinity of the branch, indicating that the structures of these two models differ at this site. Diethyl pyrocarbonate also reveals a difference in the major grooves. Comparison of the scission patterns of JA and JP by the reactive chemical probes methidium-propyl-EDTA..Fe(II), [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2,[(OP)2Cu(I)], indicates that in both cases the branch point is a site of enhanced binding for drugs, as it is in the untethered four-arm junction containing the same core sequence at the branch.

Energetic contribution of solvent-exposed ion pairs to alpha-helix structure.

Understanding the role of amino acid side-chain interactions in forming secondary structure in proteins is useful for deciphering how proteins fold and for predicting folded structures of proteins from their sequence. Analysis of the secondary structure as a function of pH in two designed synthetic peptides with identical composition but different sequences, affords a quantitative estimate of the free energy contribution of a single ion pair to the stability of an isolated alpha-helix. One peptide contains repeated blocks of Glu4Lys4. The second has repeated blocks of Glu2Lys2. The former contains significant helical structure at neutral pH while the latter has none, based on ultraviolet light circular dichroism measurements and 1H nuclear magnetic resonance spectroscopy. The difference is attributed to formation of helix-stabilizing salt-bridges between Glu- and Lys+ spaced at i, i + 4 intervals in the former peptide. The free energy of formation of a single Glu(-)-Lys+ salt-bridge can be evaluated by using a statistical model of the helix-coil transition that explicitly includes salt-bridges: the result is -0.50(+/- 0.05) kcal/mol at 4 degrees C and neutral pH in 10 mM salt, in agreement with a value derived for a single salt-bridge in a helix on the surface of a globular protein.

Helix capping in the GCN4 leucine zipper.

Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure. We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper. The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer. The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments. A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state. These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein.

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives.

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.

Stabilization of myoglobin by multiple alanine substitutions in helical positions.

We have carried out a series of multiple Xaa-->Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.

Structural analysis of the N- and C-termini in a peptide with consensus sequence.

We present a structural analysis of a peptide, the sequence of which includes amino acids that show preferences for specific positions near the N- and C-termini in protein helices. This peptide has the sequence ac-YMSEDELKAAEAAFKRHGVP-amide, which includes a strong version of an N-terminal Harper-Rose capping box structure as well as a Gly located close to the C-terminus designed to elucidate its role in C-terminal capping. The sequence of five residues at the middle is inserted to separate effects at the two ends via a helix-stabilizing linker. Application of a simulated annealing procedure using interproton distance constraints derived from 1H NOESY experiments in water reveals the presence of a C-terminal structure in this model. The C-terminus forms a folded back structure in a significant fraction of structures generated by the annealing, in most of which Gly assumes an alpha L conformation. This structure occurs within a highly flexible region of the molecule and hence is occupied only a fraction of the time.

Surface salt bridges stabilize the GCN4 leucine zipper.

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.

Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability.

The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.

Binding isotherms and the interaction between proflavin and a DNA of high G-C content.

Binding isotherms corresponding to several situations of ligand binding to a linear polymer are calculated, including cases of cooperativity or anticooperativity between the bound ligand states, multiple binding modes that are competitive or non competitive, and possible exclusive of an arbitrary number of adjacent sites upon occupancy of a site by a single ligand. The sequence generating function method of Lifson and Bradley is used, requiring the assumption that no end effects are involved. The case of strong binding of the dye proflavin to a DNA of high G-C content, that of M. lysodeikticus, is considered in detail, and a single model capable of reconciling the available kinetic and equilibrium data on this system, involving two competing binding modes, is discussed.

Structure and architecture of the bacterial virus fd. An infrared linear dichroism study.

Oriented gels of intact bacterial virus fd have been invetigated by infrared linear dichroism. Infrared absorption band maxima and dichroism indicate an alpha-helix content of the major coat protein of 95-100%. The alpha-helical rods of the coat protein are aligned parallel to the long axis of the virion with an inclination roughly estimated to approximately 37 degree. The presence of DNA infrared bands at 968, 885, 830 and 799 cm-1, the absence of a band at 860 cm-1 and the perpendicular polarization of the symmetric PO-2 stretching vibration at 1085 cm-1 are all indicative of a B-type backbone conformation in the single-stranded DNA. We find no evidence for specific interaction between aromatic side groups (phenylalanine, tyrosine) and the DNA bases. Our results independently confirm most features of the model of Marvin and co-workers [2,15 ] based on low-resolution X-ray diffraction studies. However, our findings contradict their suggestion of an A-type DNA in the bacterial virus fd. Two results are consistent with rigid and stable order in the virus. First, over a 4-day period, 65% of the peptide hydrogens remain unexchanged with deuterium. Second, changes in the relative humidity of the sample do not result in any shifts in the DNA spectrum that are characteristic of free DNA.

Structure of P401 (mast cell degranulating peptide) in solution.

P401 (also known as mast cell degranulating protein, MCD) is a minor component of honeybee venom. Its primary structure is related to that of apamin. We have studied the structure of P401 in solution by high-resolution two-dimensional 1H-NMR spectroscopy. Almost all the backbone proton resonances have been assigned by sequential assignment strategy. Analysis of NOEs shows that P401 has a conformation very similar to that of apamin. N-terminal residues Ile-1-Cys-5 are in an extended conformation and residues His-13-Asn-22 on the C-terminus are in an alpha-helical structure. These two secondary structural elements are connected by two tight turns.

Mapping tRNA and 5S RNA tertiary structures by charge dependent Fe(II)-catalyzed cleavage.

Chemical and enzymatic footprinting experiments have made it possible to identify protein binding sites in DNA and RNA, and to localize structural differences within nucleic acids to a resolution of a single base pair. We show here that by combining three reagents, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, differential maps of sites in RNA that vary in their local conformation and/or charge can be constructed. Comparison of profiles with respect to controls in the absence of a counterion such as Mg2+ allows analysis of sites responsive to tertiary structure. A single site that is labile to metals such as Pb2+ exists in tRNA(Phe) and a number of other tRNA's; this site is hyper-reactive to Fe(II), but not to the other probes. Scission induced by the neutral complex, Fe(II).EDDA, offers the most general measure of surface accessibility, since its distribution about the target molecule is insensitive to charge. Enhanced cleavage by Fe(II) relative to the other agents is detected at several adjacent sites in 5S RNA, consistent with conformational mobility. Protection at a series of positions in the arm formed by loops E and D with helix IV suggests further that at low temperature this arm interacts with loop A and helix I.

Site-specific interaction of the antitumor antibiotic dynemicin with branched DNA molecules.

A specific interaction of stable branched DNA molecules with the antitumor antibiotic dynemicin is reported. Dynemicin contains an anthraquinone and an enediyne unit, and belongs to the family of enediyne antitumor agents. DNA strand scission by dynemicin appears to involve interaction of the anthraquinone core with DNA and release of a phenyl diradical from the enediyne core that can abstract hydrogen atoms from the sugar phosphate backbone of DNA. The cleavage patterns of each labeled strand in two branched tetramers of four 16-mers are compared with those of the same strands in unbranched duplex controls. Differences between the profiles corresponding to scission of branched and duplex DNA molecules can be detected in most of the strands. The strongest differences define a specific site flanking the branch in each of two branched structures. At 18 degrees C, cleavage at strand positions demarcating the site of enhanced affinity in both junctions is observed to be 70-100% more efficient than at the corresponding sequence positions in the control duplex DNA molecules. The patterns of preferential cleavage at these sites are significantly altered in the presence of excess propidium diiodide, an intercalative drug.

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.

HMG box proteins interact with multiple tandemly repeated (GCC)n (GGC)m DNA sequences.

A number of tandemly repeated DNA sequences have the ability to form hairpin structures by forming non-standard base pairs. When (GCC)15 and (GGC)15 strands are annealed together, the expected duplex is the only product. However, when (GCC)15 is annealed with (GCC)10, depending on the relative concentrations, up to five complexes can be detected in native gels. Three of these species are susceptible to limited digestion by Exo VII, suggesting they are duplexes containing single stranded tails. The remaining two bands are resistant to the enzyme, and have low mobility on native gels, consistent with branched structures. The latter complexes bind HMG box proteins, members of a highly abundant class of non-histone proteins of the nucleus. These proteins, modeled in this study by the second box fragment from rat HMG1, HMGb, interact strongly with branched or chemically modified DNA, relative to normal duplexes. The expansion of triplet repeats in genomic DNA is associated with tumor formation as well with a variety of heritable neurologiocal disorders. It is our thesis that the stability of branched intermediate structures that arise in replication of these sequences and promote expansion can be influenced directly by the presence of two highly abundant proteins in the cell nucleus: the HMG box proteins, HMG1/2, and the histone H1, which associates with HMG1/2.