NMR and CD data have previously shown the formation of the T(4) tetraloop hairpin in aqueous solutions, as well as the possibility of the B-to-Z transition in its stem in high salt concentration conditions. It has been shown that the stem B-to-Z transition in T(4) hairpins leads to S (south)- to N (north)-type conformational changes in the loop sugars, as well as anti to syn orientations in the loop bases. In this article, we have compared by means of UV absorption, CD, Raman, and Fourier transform infrared (FTIR), the thermodynamic and structural properties of the T(4) and A(4) tetraloop hairpins formed in 5'-d(CGCGCG-TTTT-CGCGCG)-3' and 5'-d(CGCGCG-AAAA-CGCGCG)-3', respectively. In presence of 5M NaClO(4), a complete B-to-Z transition of the stems is first proved by CD spectra. UV melting profiles are consistent with a higher thermal stability of the T(4) hairpin compared to the A(4) hairpin. Order-to-disorder transition of both hairpins has also been analyzed by means of Raman spectra recorded as a function of temperature. A clear Z-to-B transition of the stem has been confirmed in the T(4) hairpin, and not in the A(4) hairpin. With a right-handed stem, Raman and FTIR spectra have confirmed the C2'-endo/anti conformation for all the T(4) loop nucleosides. With a left-handed stem, a part of the T(4) loop sugars adopt a N-type (C3'-endo) conformation, and the C3'-endo/syn conformation seems to be the preferred one for the dA residues involved in the A(4) tetraloop.
CUUG loop is one of the most frequently occurring tetraloops in bacterial 16S rRNA. This tetraloop has a high thermodynamic stability as proved by previous UV absorption and NMR experiments. Here, we present our results concerning the thermodynamic and structural features of the 10mer 5′-r(GCG-CUUG-CGC)-3′, forming a highly stable CUUG tetraloop hairpin in aqueous solution, by means of several optical techniques (UV and FT-IR absorption, Raman scattering). UV melting profile of this decamer provides a high melting temperature (60.7°C). A set of Raman spectra recorded at different temperatures allowed us to analyze the order-to-disorder (hairpin-to-random coil) transition. Assignment of vibrational markers led us to confirm the particular nucleoside conformation, and to get information on the base stacking and base pairing in the hairpin structure. Moreover, comparison of the data obtained from two highly stable CUUG and UUCG tetraloops containing the same nucleotides but in a different order permitted an overall discussion of their structural features on the basis of Raman marker evidences.
We present an experimental and theoretical investigation of the vibrational spectra of cytosine and protonated cytosine. In addition, we study spectra of cytosine and protonated cytosine with the mass of N1−H hydrogen changed to the mass of the methyl group. In this way the spectra of the heterocyclic part of cytidine are simulated. Spectral interpretation is based on the double harmonic approximation and scaled quantum mechanical (SQM) methodology. The scale factors of the ab initio HF/6-31G* and density functional Becke3-LYP/6-31G* force fields are adjusted using the frequencies and intensities measured for crystalline samples of anhydrous cytosine, cytosine monohydrate, and cytosine hydrochloride of known crystal structures. The same sets of scale factors are used for interpretation of vibrational spectra of both the neutral and protonated cytosine. These scale factors are also recommended for the spectral calculations of other nucleic acid bases and oligonucleotides. The consistency in transferring scale factors among different molecules is improved by using ring stretching scale factors that are linearly decreasing with the magnitude of the respective force constant. The original IR and Raman spectra of neutral and acidic aqueous solutions of cytosine are compared to the spectra of crystalline samples and to the calculated spectra. It is shown that frequency deviations caused by molecular environment and deviations between calculated spectra and spectra of aqueous solutions are of the same magnitude. Larger uncertainties that remain for wagging and torsional vibrations of the cytosine amino group, due to its largely anharmonic character, are significantly decreased upon cytosine protonation, which makes the cytosine amino group planar and more stiff in its out-of-plane degrees of freedom. The relative stabilities of tautomers of protonated cytosine in the gas phase and in a polar solvent are investigated at the ab initio level of theory with methods involving Hartree−Fock, MP2, MP4, and polarizable continuum approximations. In addition, discrete description of aqueous solvation was employed by using an empirical Langevine dipole model. According to these calculations, O2-protonated (enol) cytosine should slightly prevail in the gas phase, whereas in aqueous solution the N3-protonated form of cytosine is 3 ± 1 kcal/mol more stable than the enol tautomer. The energetic grounds, as well as our analysis of the vibrational spectra, support the recent finding of Purrello et al. (J. Am. Chem. Soc. 1993, 115, 760) that the enol form of protonated cytosine, in the form of cytidine monophosphate, is present in small amounts in acidic aqueous solution.
Raman and Raman optical activity (ROA) spectra of several oligo- and poly-L-proline samples of various chain lengths were measured in a wide frequency range between 120 and 1800 cm −1 and analysed with respect to the main peptide chain conformation. Specifically, formation of polyproline II (PPII) helical conformation was studied in dependence on the increasing chain length N of the (L-proline) N sample. Due to high sensitivity of the ROA technique to the conformational stability and rigidity of peptide chain we were able to determine the characteristic spectral peaks associated with formation of stable PPII helical conformation in studied systems. The most relevant peaks are located at 405, 535 and 945 cm −1 . Additionally, based on our data analysis, we were able to determine the minimal length of (L-proline) N chain necessary for creation of the stable PPII conformation as N = 6.
Raman analysis of Na+,K(+)-ATPase structural changes induced by cation binding reveals a slight decrease ( < 10%) of the alpha-helical content upon E1-E2 transition. Pronounced conformational changes of the enzyme are unlikely as the character of the environment of tyrosine residues remains unaltered. However, local changes can take place as evidenced by changes in tryptophan vibration at about 880 cm-1.
Fine effects of the hydration, charge, and conformational structural changes in l-alanyl-l-alanine (Ala-Ala) dipeptide were studied with the aid of Raman and Raman optical activity (ROA) spectra. The spectra were recorded experimentally and analyzed by means of density functional computations. A 15N and 13C isotopically labeled analogue was synthesized and used to verify the vibrational mode assignment. Calculated shifts in vibrational frequencies for isotopically labeled molecule agreed well with the experiment. The assignment made it possible to scale computed vibrational frequencies and extract better structural information from the intensities. Solvent modeling with clusters obtained from molecular dynamics led to a qualitatively correct inhomogeneous broadening of Raman spectral lines but did not bring a convincing improvement of ROA signal when compared to a standard dielectric solvent correction. In comparison with the zwitterionic form, charged anionic and cationic dipeptides provided spectral variations that indicated different conformational behavior. Only minor backbone conformational change occurs in the cation, whereas the results indicate the presence of more anion conformers differing in the rotation of the NH2 group and the backbone ψ-angle. These findings are in agreement with previous electronic circular dichroism (ECD) and NMR studies. The results confirm the large potential of the ROA technique for the determination of final details in molecular structure and conformation.
CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with Kd ∼54 μM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-d-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-d-galactosamine. Site-directed mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.
