Carbon-13 nuclear magnetic resonance studies of polyamino acids: the helix-coil transition of poly-L-lysine

The helix-coil transition of poly-l-lysine hydrochloride ((Lys)_n) in aqueous solution has been studied by ¹³C Fourier-transform nuclear magnetic resonance spectroscopy. As reference compounds dodeca-l-lysine hydrobromide ((Lys)^?₁₂, tri-l-lysine hydrochloride ((Lys)₃), and l-lysine hydrochloride (Lys), have been also studied by the same method. It is found that ¹³C spin-lattice relaxation times t₁ of the carbonyl and the side-chain carbons decrease sharply at pD 10.2 which is the midpoint of the transition from the random-coil to the α-helix. Similarly the T₁ values of the carbonyl groups of (Lys)^?₁₂ decrease at this point in a more moderate way, while no change is observed for those of the side-chain carbons. This is interpreted in terms of the reduced α-helicity involved for (Lys)^?₁₂.The variation of ¹³C chemical shifts with pD for (Lys)_n and (Lys)^?₁₂ show the same trend:downfield shifts at higher pD. Furthermore, nonterminal and C-terminal residues of (Lys)₃ show similar behavior. Thus it is concluded that the ¹³C chemical shift changes are caused mainly by the pD changes and not by the conformational transition. Conversion from α-helix to β-structure by elevation of temperature at pD 11.2 results in narrowing and downfield shifts of the ¹³C resonances of (Lys)_n.     

Proton magnetic resonance study of the binding of purine to polyuridylic acid

The interaction of unsubstituted purine with polyuridylic acid in D₂O solution at neutral pD has been studied by high resolution proton magnetic resonance spectroscopy. The poly U proton resonances were shifted to higher fields by the added purine, indicating that purine binds to the uracil bases of the polymer by base stacking. Severe broadening of the purine proton resonances was also observed, providing strong evidence for the intercalation of purine between adjacent uracil bases of the polymer. The line widths of the poly U proton resonances were not noticeably broadened in the presence of purine; thus, the binding of purine to poly U does not result in a more rigid or ordered structure for the polymer.     

Calorimetric investigation of the helix-coil conversion of polyuridylic acid

The heats of the conformational conversion ΔH_c of polyuridylic acid (polyU) in the presence of various cations were determined using a differential scanning microcalorimeter and an isothermal mixing calorimeter. The first method yields a value of ΔH_c = 5.2 kcal/mol of base pairs, the second one a value of ΔH_c = 6.4 kcal/mol of base pairs. Knowing ΔH_c and the maximal slope of the degree of conversion, which was determined from the temperature dependence of the absorbance of polyU solutions at 260 nm, we were able to evaluate the parameter of cooperativity σ. From the relation σ = e the stacking free energy ΔF_stack could be calculated. Dividing the apparent enthalpy of conversion by the calorimetrically measured enthalpy yields the cooperative length N₀ of one helical segment at the midpoint of conversion. The results show that the type of the cation has no influence on the magnitude of ΔH_c, whereas the cooperativity of polyU is influenced by the various cations.     

Steady-state carbon-13 nuclear magnetic resonance spectra of acyl-alpha-chymotrypsin

When [13C]carbonyl-enriched p-nitrophenyl 5-n-propyl-2-furoate is incubated with alpha-chymotrypsin, a new peak appears in the 13C NMR spectrum. On the basis of its position and the fact that it is "chased" with unlabeled substrate, we conclude that this new signal is due to the acyl-enzyme intermediate. In spectra taken during steady-state turnover, the acyl-enzyme ester carbonyl 13C chemical shift displays a pH dependence that fits to a titration curve with an apparent pK of 7.1 (0.1). The apparent pK of the kcat vs. pH curve for enzyme-catalyzed hydrolysis of the same substrate under conditions differing only in reactant concentration is 7.0 (0.1). We have found no spectral evidence for a tetrahedral intermediate.     

Interactions of long-chain aldehydes with luciferase. A carbon-13 nuclear magnetic resonance study.

The interaction of long-chain aldehydes with bacterial luciferase has been studied by 13C NMR spectroscopy of natural-abundance and 13C-enriched 1-dodecanal. At high substrate/enzyme ratios, the spin-spin relaxation rates of C(1)-C(3) are faster than for the other carbons and are in the order C(1) greater than C(2) greater than C(3). The aldehyde is strongly bound in the active site along the entire length of the alkyl chain with the strongest interaction at the CHO group. At low substrate/enzyme ratios, interactions are apparent at C(10), which are removed upon denaturation of the enzyme. Spin-spin and spin-lattice relaxation rates were measured for odd-carbon 13C-enriched 1-dodecanal in the presence of luciferase. From the ratios of T1/T2 a single value of (1.8 +/- 0.7) X 10(-8) s was calculated for the rotational correlation time tc for the complex.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

Three-stranded helix-coil equilibrium in polyuridylic acid-adenosine complex

Interaction of poly U (polyuridylic acid) and adenosine is studied by following the changes in ultraviolet absorbance in the wavelength region near the isochromic wave-length for the complex formation. The interaction is studied as a function of temperature, concentration of adenosine, and ionic strength, while the concentration of poly U was held constant. It is confirmed that only the three-stranded complex with the stoichiometry 1A to 2U is formed and that it dissociates directly into free poly U and adenosine. No discontinuity of any kind was apparent in the melting curves, and poly U was found to possess no ordered structure above 10°C under the conditions used. The results were, therefore, analyzed in terms of an exact helix–coil equilibrium theory using the mismatching model, i.e., assuming that either completely formed base triplet or completely free unbonded bases only exist, and that the two sections of the polymer chains forming closed loops need not contain the same number of unbonded bases. Self-association of free adenosine was taken into consideration. (Base triplet is analog of base pair for a three-stranded helical complex. It refers to a unit of three coplanar bases, in this case two uracils and one adenine, hydrogen bonded to one another to form a triplet. Such triplets may stack over one another along the helical axis, and when they are so stacked the bases of two triplets next to each other may have stacking interactions between them.) The values for enthalpy and entropy changes, both per mole of base triplets, were obtained for the following processes at neutral pH and moderate to high salt concentrations. (1) Growfh: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form a base triplet next to an already formed base triplet with which it has stacking interactions, a process that involves both hydrogen bonding and base stacking interactions, ΔH_s, = ?19 ± 2 kcal, ΔS_s = ?55 ± 6 clausius; (2) Initiation: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form an isolated base triplet, a process that involves only hydrogen bonding interactions, ΔH_b* = 4.5 ± 2 kcal, ΔS_b* = 6.6 ± 3 clausius; and (3)Interruption: Unstacking of two stacked base triplets initially next to each other by formation of an interruption (viz. a closed loop) between them, a process that involves only base stacking interactions, ΔH_b = 23.5 ± 3 kcal, ΔS_b = 61.6 ± 7 clausius, where the entropy changes include contributions other than the configurational entropy of closed loops. The discrepancy between our results and the calorimetric ΔH_s of ?13 kcal is attributed to (i) the possible effects of salt arid polymer on the self-association of free adenosine, (ii) the uncertainty in the value of the parameter for the probability of ring closure, and (iii) the contributions due to the partial molal enthalpy of the solvent and the unstacking of any poly U structure to the calorimetric enthalpy.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

The carbon-13 nuclear magnetic resonance spectra of four eudesmane sesquiterpenols

The ¹³C NMR spectra of geosmin, selina-4(14),7(11)-diene-99-ol and two dihydroeudesmol isomers have been obtained and the individual resonances assigned. Several different empirical correlations developed by others have been combined in simple calculations to predict chemical shift values for sesquiterpenols of the eudesmane group.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.