NMR RELAXATION STUDIES OF FAST INTERNAL MOTIONS IN NUCLEIC ACIDS
Lukáš Trantírek, Erik Caha and Radovan Fiala
Investigations of intramolecular dynamics on the nanosecond to picosecond time scale by solution NMR spectroscopy are based on the spin relaxation properties of nuclei such as 15N or 13C. The most commonly measured quantities are the longitudinal relaxation rate constant, R1, the transverse relaxation rate constant, R2, and the steady state nuclear Overhauser effect (NOE). The values of these experimentally obtained parameters can be expressed as linear combinations of spectral density functions. The spectral density function characterizes the overall rotational diffusion of the molecule as well as its intramolecular motions. Two approaches exist for the interpretation of the experimental data. In spectral density mapping, the values of the spectral density function J(ω) at characteristic frequencies are determined from the relaxation data . The "model-free" formalism assumes a particular form of the spectral density function, whose parameters then characterize amplitudes and time scales of the molecular motions .
The methods for measuring 15N relaxation parameters have been well established thanks to a large body of literature on protein dynamics studied through amide nitrogen relaxation. Because of the similarity of the spin environments, the procedures developed for protein amide nitrogen are directly applicable to imino nitrogen of guanine and uracil . However, 15N relaxation study of nucleic acids can yield the dynamic properties of guanine and uracil bases only. For a more complete picture, the use of 13C relaxation data is highly desirable. The most suitable candidates for relaxation studies in nucleic acids are C8 carbons of purines, C6 carbons of pyrimidines and C1' of the sugar.
In order to bring insight into the internal dynamics of RNA tetraloops we have performed a 13C NMR relaxation and molecular dynamics study of 14-nt RNA hairpins GGCACUUCGGUGCC and GGCACGCAAGUGCC (the underlined nucleotides form the loops). The UNCG and GNRA families of stable RNA hairpins (where N is any nucleotide and R is purine) have very similar overall folds. However, the biological roles of these two sequences appear different. The differences have been attributed to distinct dynamical properties of the two sequences .
We have measured R1 and R1ρ relaxation rates for C8 of purines, C2 of adenines, C6 and C5 of pyrimidines as well as for C1' of the ribose sugars at several magnetic field strengths. The data have been interpreted in the framework of modelfree analysis characterizing the internal dynamics of the molecules by order parameters and correlation times for fast motions on the picosecond to nanosecond time scale and by contributions of chemical exchange.
While both tetraloops exhibit increased mobility on the fast time scales, with the GCAA loop we have detected a significant contribution of conformational dynamics on the millisecond to microsecond time scale. This is consistent with the observations that the GNRA family appears more flexible and tolerant of the conformational changes important for molecular recognition.
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