An affinity scale for the interaction of amino acid residues with nucleic acids

The affinity of amino acid residues to nucleic acids is probed by measurements of melting temperatures t_m for the helix–coil transition at various concentrations of amino acid amides. The increase of t_m on addition of ligand is described by the equation t_m = t*_m + αlog(1+K_tc_λ), where t*_m is the melting temperature in the absence of ligand, c_λ the ligand concentration, and K_t the “t_m-onset” constant, which is analogous to an equilibrium constant. It is shown that K_t is closely related to the affinity of the ligands to the double helix, whereas the slope α mainly reflects the preference of the ligand binding to the helix versus the coil form. In the case of the amino acid amides, α is found to be virtually independent of the nature of the side chain with few exceptions, e. g., aromatic amides. The t_m-onset constant, however, strongly depends on the nature of the amino acid side chain. For simple aliphatic amino acids, the relative free energy of binding decreases with increasing hydrophobic free energy, e.g., a high affinity is found for Gly-amide and a low affinity for Leu-amide. This relation is modified by functional groups like OH in Ser-amide. The helices poly[d(A-T)], ploy[d(I-C)]. and poly[d(A-C)]·poly[d(G-T)] exhibit similar affinity scales with relatively small variations. Our results demonstrate that the hydrophilic character of double helices at their surface disfavors binding of hydrophobic ligands unless special contacts can be formed. From our results we establish an affinity scale for the binding of amino acids to double helices.     

A proton motive force transducer and its role in proton pumps, proton engines, tobacco mosaic virus assembly and hemoglobin allosterism

In this paper I am proposing a new, conformationally dependent basic site in proteins. The initial formulation of this proposal was based on the following: (1) bacteriorhodopsin is a light-driven proton pump and as such is a prototype for understanding proton-mediated energy transduction in biological systems; (2) current evidence suggests about 2 protons are pumped for each photon absorbed; (3) given the usual role of prolines as helix breakers, it is surprising to find about 2 prolines deeply embedded in the membrane-spanning, probably α-helical, portion of the bacteriorhodopsin molecule; (4) another presumptive proton translocator, the F₀ proteolipid, is also helical and has a critical proline in its structure; (5) workers interested in protein folding have explained the existence of fast and slow folding subgroups of the same protein molecule as being due to cis : trans isomerization about the proline imide group; (6) the cis : trans isomerization is acid catalyzed; (7) simple chemical considerations predict that the proton affinity of the proline nitrogen should increase dramatically as the imide group is distorted away from planarity and should be a maximum midway between the cis and trans forms; thus, stabilization of the intermediate by protonation accounts for the acid catalysis of the proline cis : trans isomerization.Linking these observations together suggests that proline-containing α-helices may play a role in proton motive energy transduction. Due to the absence of a proton on the proline nitrogen, a proline-containing helix has a “proton hole” between the proline nitrogen and the carbonyl oxygen four residues earlier in the sequence. Here I propose a model in which the paramount feature is the change in pK_a associated with a change in geometry of the “proton hole.” Order of magnitude calculations suggest that the proton hole should change its pKa by about 8 units, corresponding to a 10⁸ change in proton affinity, for every 10 kcal of distortion energy, V. Calculations also show that it is energetically feasible to modulate the pK_a of this site over the dynamic range of pK_a = 2–14. Such a large value for $\text{ΔpK}{}_{\mathrm{a}}\text{ΔV}$ and such a dynamic range makes this site an ideal basis for an “integral proton injector,” an abstract model for proton pumping suggested on purely theoretical grounds by Nagle &; Mille (J. chem. Phys.74, 1367–1372, 1981).Finally, two well studied proteins, the α-chain of hemoglobin and tobacco mossaic virus coat protein, both show features in their X-ray determined structures suggesting the possibility of protonation and deprotonation of the proton hole in α-helices containing proline. For TMV coat protein, there is a proline-containing α-helix that is located precisely in the region of the protein which undergoes an acid-induced conformational rearrangement. Structural changes at this locus have been singled out in comparisons of the X-ray structures of the TMV protein in its two conformations. For the α-chain of horse hemoglobin, there are two concurrent sites that are likely protonated and one contrary site that likely becomes deprotonated as hemoglobin converts from the liganded to the deoxy form. The contrary proline is proposed to help maintain co-operative oxygen binding over a wide pH range. The absence of one of the concurrent proline site in marsupial hemoglobin accounts for the small Bohr effect exhibited by these hemoglobins. The absence of the contrary proline site in carp hemoglobin accounts in a very logical way for the large Bohr effect and the lack of cooperative oxygen binding at both low and high pH by this hemoglobin.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

Studies of the zein-like α-prolamins based on an analysis of amino acid sequences: Implications for their evolution and three-dimensional structure

α-Prolamins are the major seed storage proteins of species of the grass tribe Andropogonea. They are unusually rich in glutamine, proline, alanine, and leucine residues and their sequences show a series of tandem repeats presumed to be the result of multiple intragenic duplication. Two new sequences of α-prolamin clones from Coix (pBCX25.12 and pBCX25.10) are compared with similar clones from maize and Sorghum in order to investigate evolutionary relationships between the repeat motifs and to propose a schematic model for their three-dimensional structure based on hydrophobic membrane-helix propensities and helical “wheels.” A scheme is proposed for the most recent events in the evolution of the central part of the molecule (repeats 3 to 8) which involves two partial intragenic duplications and in which contemporary odd-numbered and even-numbered repeats arise from common ancestors, respectively. Each pair of repeats is proposed to form an antiparallel α-helical hairpin and that the helices of the molecule as a whole are arranged on a hexagonal net. The majority of helices show six faces of alternating hydrophobic and polar residues, which give rise to intersticial holes around each helix which alternate in chemical character. The model is consistent with proteins which contain different numbers of repeats, with oligomerization and with the dense packaging of α-prolamins within the protein body of the seed endosperm. © 1993 Wiley-Liss, Inc.     

Kinetic steps for α-helix formation

The kinetics of α-helix formation in polyalanine and polyglycine eicosamers (20-mers) were examined using torsional-coordinate molecular dynamics (MD). Of one hundred fifty-five MD experiments on extended (Ala)₂₀ carried out for 0.5 ns each, 129 (83%) formed a persistent α-helix. In contrast, the extended state of (Gly)₂₀ only formed a right-handed α-helix in two of the 20 MD experiments (10%), and these helices were not as long or as persistent as those of polyalanine. These simulations show helix formation to be a competition between the rates of (a) forming local hydrogen bonds (i.e. hydrogen bonds between any residue i and its i + 2, i + 3, i + 4, or i + 5th neighbor) and (b) forming nonlocal hydrogen bonds (HBs) between residues widely separated in sequence. Local HBs grow rapidly into an α-helix; but nonlocal HBs usually retard helix formation by “trapping” the polymer in irregular, “balled-up” structures. Most trajectories formed some nonlocal HBs, sometimes as many as eight. But, for (Ala)₂₀, most of these eventually rearranged to form local HBs that lead to α-helices. A simple kinetic model describes the rate of converting nonlocal HBs into α-helices. Torsional-coordinate MD speeds folding by eliminating bond and angle degrees of freedom and reducing dynamical friction. Thus, the observed 210 ps half-life for helix formation is likely to be a lower bound on the real rate. However, we believe the sequential steps observed here mirror those of real systems. Proteins 33:343–357, 1998. © 1998 Wiley-Liss, Inc.     

Theory of protein molecule self-organization. I. Thermodynamic parameters of local secondary structures in the unfolded protein chain

This paper presents the results of a stereochemical analysis of local interactions in unfolded protein chains (sterical repulsions, hydrogen, and hydrophobic bonds, etc.) by means of space-filling modeles. On the basis of this analysis, an evaluation is made of thermodynamic parameters controlling the building-in of all the 20 natural amino acid residues in all the physically possible position of local secondary structures (α-helices, including α-helices with short fragments of helices 3₁₀ at the C-terminus; β-bends of different types, helices 3₁₀, and their combinations) as well as thermodynamic parameters of separate hydrogen bonds of polar side groups with the neighbor peptide groups (“local contacts”). The accuracy of the obtained results is discussed.     

The Stabilizing Effects of O-glycosylation on the Secondary Structural Integrity of the Designed α-loop-α motif by Molecular Dynamics Simulations

Abstract

In this study, various 400 ps molecular dynamics simulations were conducted to determine the stabilizing effect of O-glycosylation on the secondary structural integrity of the design α-loop-α motif, which has the optimal loop length of 7 Gly residues (denoted as N-A₁₆G₇A₁₆-C). In general, O-glycosylation stabilizes the structural integrity of the model peptide regardless of the length and position of glycosylation sites because it decreases the opportunity for water molecules to compete for the intramolecular hydrogen bonds. The designed peptide exhibits the highest helicity when residues 11 and 31 are replaced with Ser residues followed by O-linked with 3 galactose residues, representing the “face-to-face” glycosylation near the loop. In this case, the loop exhibits an extended conformation and several new hydrogen bonds are observed between the main chain of the loop and the galactose residues, resulting in decreasing the fluctuation and increasing the stability of the entire peptide. When the glycosylation are made close to the loop, the secondary structural integrity of the α-loop-α motif increases with the number of galactose residues. In addition, “face- to-face” glycosylation increases the structural integrity of this motif to a greater extent than “back-to-back” glycosylation. However, when the glycosylation are created away from the loop and near the N- and C-termini, no general rule is found for the stabilizing effect.     

Hydrogen Exchange of Individual Amide Protons in the E. Coli lac Repressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Abstract

Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy. The exchange rates of amide protons, stabilized by the hydrogen bonds of the three α-helices of the headpiece, could be determined quantitatively. The exchange rates in these helices showed repetitive patterns of about three to four residues. A correlation with the position of the amide proton in the interior or the exterior of the α-helix of the protein was found. The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R. et al., J. Mol. Biol. 182, 179-182 (1985)).     

The intranuclear helices of Euplotes: Their substructure,localization, and apparent migration to the cytoplasm

Study of the fine structure of the macronucleus in Euplotes eurystomus, a ciliate protozoon, during various stages of the cell division cycle has yielded new information about intranuclear helices. They are frequently observed at the periphery of chromatin bodies or next to the nuclear envelope, and they appear to be a constituent of nucleoli. The fibril that forms a helix is about 11–15 nm thick, and torus profiles of helices cut in cross section are about 35 nm in diameter. In substructure the helix is composed of a thin strand 3–5 nm thick which is coiled to form the 11–15 nm fibril; so the helix is a super-coiled structure. The intranuclear helices are present in the macronucleus throughout the cell cycle. They do not show obvious changes of relative abundance nor changes of relative localization in the nucleus, with one exception: they were never observed in the diffuse zone of replication bands. Evidence is presented indicating that nuclear helices migrate to the cytoplasm through nuclear pores. Although the chemical composition of the Euplotes intranuclear helices is unknown, information in the literature on similar helices in Amoeba indicates that they contain RNA and not DNA. The observations on Euplotes helices are consistent with a concept of “packaged” RNA for transport to the cytoplasm.     

The conformation of an alamethicin in methanol by multinuclear NMR spetroscopy and distance geometry/simulated annealing

The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. ¹H-, ¹³C-, and ¹⁵N-nmr chemical shifts and homonuclear ¹H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 3₁₀-helical conformation. The large differences in the ¹³C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc.     

Magnesium reduces affinities of antagonists at rat cortex α2-adrenergic receptors labeled with 3H-clonidine: Evidence for heterogeneity of α2-receptor conformations with respect to antagonists

³H-clonidine labeled two binding sites in rat cortex membranes with apparent K_D values of about 1.0 and 5.9 nM. These sites appeared analogous to “super-high” (SH) and “high” (H) affinity states of the α₂-receptor described in human platelets. 10 mM magnesium increased the number of SH receptors by 30% whereas 100 μM GTP reduced SH₃receptor number by 45% with no significant change in the K_D of ³H-clonidine at α₂(SH) sites. In drug competition studies using 1.0 nM ³H-clonidine, 100 μM GTP reduced the affinity of clonidine and increased the affinity of yohimbine, whereas 10 mM magnesium increased the affinity of clonidine and reduced the affinity of yohimbine. The effect of magnesium on the affinity of several antagonists at cortex ³H-clonidine sites ranged from none (phentolamine) to a 6-fold reduction (piperoxan). These data indicate that different states of the α₂-receptor exhibit different affinities for some antagonists.     

The Alacoil: A very tight,antiparallel coiled-coil of helices

The Alacoil is an antiparallel (rather than the usual parallel) coiled-coil of α-helices with Ala or another small residue in every seventh position, allowing a very close spacing of the helices (7.5–8.5 Å between local helix axes), often over four or five helical turns. It occurs in two distinct types that differ by which position of the heptad repeat is occupied by Ala and by whether the closest points on the backbone of the two helices are aligned or are offset by half a turn. The aligned, or ROP, type has Ala in position “d” of the heptad repeat, which occupies the “tip-to-tip” side of the helix contact where the Cα–Cβ bonds point toward each other. The more common offset, or ferritin, type of Alacoil has Ala in position “a” of the heptad repeat (where the Cα-Cβ bonds lie back-to-back, on the “knuckle-touch” side of the helix contact), and the backbones of the two helices are offset vertically by half a turn. In both forms, successive layers of contact have the Ala first on one and then on the other helix. The Alacoil structure has much in common with the coiled-coils of fibrous proteins or leucine zippers: both are α-helical coiled-coils, with a critical amino acid repeated every seven residues (the Leu or the Ala) and a secondary contact position in between. However, Leu zippers are between aligned, parallel helices (often identical, in dimers), whereas Alacoils are between antiparallel helices, usually offset, and much closer together. The Alacoil, then, could be considered as an “Ala anti-zipper.” Leu zippers have a classic “knobs-into-holes” packing of the Leu side chain into a diamond of four residues on the opposite helix; for Alacoils, the helices are so close together that the Ala methyl group must choose one side of the diamond and pack inside a triangle of residues on the other helix. We have used the ferritin-type Alacoil as the basis for the de novo design of a 66-residue, coiled helix hairpin called “Alacoilin.” Its sequence is: cmSP DQWDKE A AQYDAHA QE FEKKS HRNng TPEA DQYRHM A SQY QAMA QK LKAIA NQLKK Gseter (with “a” heptad positions underlined and nonhelical parts in lowercase), which we will produce and test for both stability and uniqueness of structure.     

Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å

The dibenzothiophene (DBT) monooxygenase DszC, which is the key initiating enzyme in “4S” metabolic pathway, catalyzes sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO₂). Here, we report the crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Intriguingly, two distinct conformations occur in the flexible lid loops adjacent to the active site (residue 280–295, between α9 and α10). They are named “open”' and “closed” state respectively, and might show the status of the free and ligand‐bound DszC. The molecular docking results suggest that the reduced FMN reacts with an oxygen molecule at C4a position of the isoalloxazine ring, producing the C4a‐(hydro)peroxyflavin intermediate which is stabilized by H391 and S163. H391 may contribute to the formation of the C4a‐(hydro)peroxyflavin by acting as a proton donor to the proximal peroxy oxygen, and it might also be involved in the protonation process of the C4a‐(hydro)xyflavin. Site‐directed mutagenesis study shows that mutations in the residues involved either in catalysis or in flavin or substrate‐binding result in a complete loss of enzyme activity, suggesting that the accurate positions of flavin and substrate are crucial for the enzyme activity. Proteins 2014; 82:1708–1720. © 2014 Wiley Periodicals, Inc.     

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F₁F_o-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K_0.5=0.27 mM) and low (K_0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg²⁺ saturating condition (high affinity site, K_0.5=0.10 mM, and low affinity site, K_0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.     

A Hydrogen Bonded Chain in Bactereorhodopsin by Computer Modelling Approach

Abstract

The seven α-helical segments of Bacteriorhodopsin (BR) passing through the membrane are investigated for a continuous Hydrogen Bonded Chain (HBC). The study is carried out by computer modelling approach. It is assumed that the seven helices are placed as (AGFEDCB), which has been accepted as the best model by several groups. Helices A, D, E and G are considered to be present in right handed α-helical conformation. The inter-orientation of these helices are represented by Eulerian angles α, β and γ. For the helices B, C and F which contain Proline in the middle, several conformational possibilities were considered. In these cases apart from the Eulerian angles α, β and γ, the dihedral angles φ_{p_1} and ψ_{p_1} of the residues that are succeeded by Proline residue in the helical regions were also used in fixing the position of the helices with respect to each other. All these parameters were varied to fit with the top, middle and bottom distances reported by electron diffraction studies. Good fit was obtained for all right handed α-helical conformations and also for helices B, C and F with a left handed turn at the residue preceeding proline. Hence two structures were analysed for continuous HBC, Structure I which contained all the seven helices in right handed α-helical conformation and Structure II, which had the helices A, D, E and G in right handed conformation and the helices B, C and F in right handed α-helical conformation with a left handed turn at the residue preceeding proline. All possible staggered conformations were considered for the side chains and the inter atomic distances were analysed for Hydrogen bonds. It was possible to obtain a continuous chain in both the structures which includes most of the residues found to be important by the experiments. However Lys-216 has to be considered in two different conformations to connect the cytoplasmic side with the extra cellular side. The overall height spanned by HBC is about 25Å. The chains obtained by both the structures I and II are analysed in terms of the conformational parameters. It has also been possible to place the retinal in the region as predicted by the experiments. The Tryptophan residues which affect the spectral characterestics can be aligned on either side of the retinal.     

The effects of chain length on the secondary structure of oligoadenylates

The oligoadenylates (Ap)_2–4A have been studied by proton magnetic resonance (pmr) spectroscopy. All the exterior base protons and a number of the interior base proton resonance have been assigned. The results of this work showed that the adenine bases in these oligoadenylates are intramolecularly stacked at 20°C with their bases oriented preferentially in the anti conformation about their respective glycosidic bonds. The oligomers were found to associate extensively even at concentrations of 0.02 M, primarily via “end-to-end” stacking. With increasing temperature, the oligomer bases destack, but it is argued that this unfolding process cannot be described in terms of a two-state stacked versus unstacked model. Instead, the temperature dependences of the base proton chemical shifts support a base-oscillation model. The relationship between this model and the two-state model is discussed. Finally, on the basis of the chain-length dependence of the proton chemical shifts of the various adenine bases, it was concluded that subtle variations in the secondary structure of these oligomers exist with increasing chain length. Evidence is presented to show that the effects of distant base shielding are considerably smaller than what was previously estimated. The observed departures from the “extended dimer” model are attributed to differences in the relative orientations of the bases with respect to their neighbors in the oligomer.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady‐state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47–71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C‐terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the k_cat value unchanged; however, removing three or more residues reduces k_cat by 1000‐fold. In contrast, the glucose K_0.5 and K_D values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline‐66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK‐I) eliminates cooperativity without impacting the k_cat and glucose K_0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.     

Cooperative nonenzymic base recognition II. thermodynamics of the helix-coil transition of oligoadenylic + oligouridylic acids

The properties of oligonucleotide helices of adeuylic- and uridylic acid oligomers have been investigated by measurements of hypo-and hyperchromieity. High ionic strengths favor the formation of triple helices. Thus, the double helix-coil transition can be studied (without interference by triple helices) only at low ionic-strength. A “phase diagram” is given representing the T_m-values of the various transitions at different ionic strengths for the system A(pA)₁₇ + U(pU)₁₇. Oligonucleolides of chain lengths <8 always form both double and triple helices at the nucleotide concentrations required for base pairing. For this reason the double helix-coil transition without coupling of the triple helix equilibrium can only be measured for chain lengths higher than 7. Melting curves corresponding to this transition have been determined for chain lengths 8, 9, 10, 11, 14 and 18 at different concentrations. An increase in nucleotide concentration leads to an increase in melting temperature. The shorter the chain length the lower the T_m-value and the broader the helix-coil transition. The experimental transition curves have been analysed according to a staggering zipper model with consideration of the stacking of the adeuylic acid single strands and the electrostatic repulsion of tlip phosphate charges on opposite strands. The temperature dependence of the nucleation parameter has been accounted for by a slacking factor x. The stacking factor expresses the magnitude of the stacking enthalpy. By curve fitting xwas computed to be 0.7, corresponding to a stacking enthalpy of about S kcal/mole. The model described allows the reproduction of the experimental transition curves with relatively high accuracy. In an appendix the thermodynamic parameters of the stacking equilibrium of poly A and of the helix-coil equilibria of poly A + poly U at neutral pH are calculated (ΔH_A = ?7.9 kcal/mole for the poly A stacking and ΔH₁₂ = ?10.9 kcal/mole for the formation of the double helix from the randomly coiled single strands). A formula for the configurational entropy of polymers derived by Flory on the basis of a liquid lattice model is adapted to calculate the stacking entropies of adenylic oligomers.     

An X-ray diffraction study of poly(L-lysine hydrobromide)

A structural study of the synthetic polypeptide poly(L -lysine hydrobromide) has been made by X-ray fiber techniques. The investigation was undertaken to determine whelther this polymer undergoes conformational transitions as a function of hydration in a manner similar to other chemically related basic polypeptides. Specifically, a comparison with the previously reported structures of the hydrochloride form of poly(L -lysine) was sought. Homogeneous powder mixtures with various amounts of water and oriented fibers of poly(L -lysine hydrobromide) at different relative humidities were X-ray photographed. Reversible transitions amorphous state ? β-pleated sheet ? α-helix ? isotropic solution as a function of increasing/decreasing degrees of hydration were found. The β-pleated-sheet conformation was observed between 33% and 76% relative humidities (containing about one and three molecules of water per residue, respectively). Each pleated sheet was formed by “antiparallel” chains, and the sheets were piled up along the b-axis. The spacings of this conformation did not vary appreciably with hydration. The observed reflections at 52% relative humidity (1.4 molecules of water per residue) could be indexed satisfactorily in terms of an orthorhombic unit cell, of space group P2₁22₁, with a = 9.52 Å, b = 16.44 Å, and c = 6.80 Å. These dimensions were shown by models to be compatible with the proposed structure. The α-helix conformation was present in specimens photographed at 76% relative humidity and up, and containing between three and fifteen molecules of water per residue. The helices were packed parallel to each other in a hexagonal array but randomly along or about their lengths. Increasing the hydration from five to fifteen molecules of water per residue causes the a-axis to increase from 16.9 to 20.8 Å. Twenty molecules of water per residue produced an isotropic solution. Despite some structural differences between the hydrobromide and hydrochloride forms it is concluded that the role played by the anions is mainly related to determining the water content levels at which conformational changes occur. Therefore, the anions do not significantly influence the prevailing conformation in this particular system, but might affect the packing arrangement of the polypeptide chains.