Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using ¹⁵N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R ₂ values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T ₁ values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R ₂/R ₁ is anisotropic (D _par/D _perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with ¹⁵N relaxation rates and the estimation of R _ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.     

Direct measurement of the 15N CSA/dipolar relaxation interference from coupled HSQC spectra

Here we propose a method for the measurement of the ¹⁵N CSA/dipolar relaxation interference based on direct comparison of the ¹⁵N doublet components observed in a ¹H-coupled ¹H-¹⁵N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the ¹⁵N CSA/dipolar cross-correlation rates.     

Task Relevance Enhances Early Transient and Late Slow-Wave Activity of Distributed Cortical Sources

The primary purpose of these studies was to link together concepts related to attention/working memory and feedforward/feedback activity using MEG response profiles obtained in humans. Similar to recent studies of attention in monkeys, we show early spike-like activity (<200 ms poststimulus), most likely reflecting an early transient excitatory response mixed with feedback influences, followed by slow-wave activity (>200 ms poststimulus) in MEG cortical response profiles evoked by a visual working memory task. We experimentally dissociated the early transient activity from the later sustained activity (predominately feedback) by conducting an auditory size classification task. Words, representing common objects, evoked activity in occipital cortex (presumably due to imagery) even though visual stimuli were not present in this task. The initial spike was absent from the response profile obtained from occipital cortex, leaving only slow-wave activity, thereby allowing us to characterize or profile feedback activity in this situation. Attention or task relevance enhanced the initial spike and slow-wave activity in visually responsive areas. Prefrontal activity, along the superior frontal sulcus, evoked by the working memory task, was active later in time than initial activity in visual cortex and later than the earliest effect of attention modulation in visual cortex.     

Local mobility of 15N labeled biomolecules characterized through cross-correlation rates: Applications to paramagnetic proteins*

The mobility of 15N labeled proteins can be characterized by measuring the cross-correlation rates N,NI that govern the conversion of Zeeman order Nz of an amide 15N nucleus into longitudinal two-spin order 2NzIz involving the amide 15N and 1H nuclei. This represents an alternative to the measurement of 15N self-relaxation rates 1/T1 and 1/T2 or 1/T1. The rate of interconversion between Nz and 2NzIz is due to cross-correlation between fluctuations of different interactions and is not affected by a variety of relaxation mechanisms that contribute to the self-relaxation rates 1/T1, 1/T2 and 1/T1. Spin diffusion among protons, which affects the measurements, can be quenched by various means that are evaluated by experiments and simulations. By applying an off-resonance radio-frequency (RF) field in the vicinity of the nitrogen resonance, the spectral density function J() can be determined at the frequency origin and at the nitrogen Larmor frequency. The methods are applied to the paramagnetic High-Potential Iron-Sulfur Protein iso I (HiPIP I) from E. halophila in its reduced state.     

Spin-state selection for increased confidence in cross-correlation rates measurements

A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual ¹⁵N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S³E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92–97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7562-8     

Determination of <Superscript>13</Superscript>C CSA Tensors: Extension of the Model-independent Approach to an RNA Kissing Complex Undergoing Anisotropic Rotational Diffusion in Solution

Chemical shift anisotropy (CSA) tensor parameters have been determined for the protonated carbons of the purine bases in an RNA kissing complex in solution by extending the model-independent approach [Fushman, D., Cowburn, D. (1998) J. Am. Chem. Soc. 120, 7109–7110]. A strategy for determining CSA tensor parameters of heteronuclei in isolated X–H two-spin systems (X = ¹³C or ¹⁵N) in molecules undergoing anisotropic rotational diffusion is presented. The original method relies on the fact that the ratio κ₂=R₂^auto/R₂^cross of the transverse auto- and cross-correlated relaxation rates involving the X CSA and the X–H dipolar interaction is independent of parameters related to molecular motion, provided rotational diffusion is isotropic. However, if the overall motion is anisotropic κ₂ depends on the anisotropy D_||/D_⊥ of rotational diffusion. In this paper, the field dependence of both κ₂ and its longitudinal counterpart κ₁=R₁^auto/R₁^cross are determined. For anisotropic rotational diffusion, our calculations show that the average κ_av = 1/2 (κ₁+κ₂), of the ratios is largely independent of the anisotropy parameter D_||/D_⊥. The field dependence of the average ratio κ_av may thus be utilized to determine CSA tensor parameters by a generalized model-independent approach in the case of molecules with an overall motion described by an axially symmetric rotational diffusion tensor.     

Motional dynamics of residues in a beta-hairpin peptide measured by 13C-NMR relaxation.

Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. 13C-NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, R1GITVNG7KTYGR12, has been shown to partially fold in a relatively stable beta-hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G11, were selectively 13C-enriched, and 13C-NMR relaxation experiments were performed to obtain auto- and cross-correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coefficients. Our results indicate that, of the three glycines, G7 within the hairpin beta-turn displays the most correlated phi(t),psi(t) rotations with its axis of rotation bisecting the angle defined by the H-C-H bonds. These positively correlated bond rotations give rise to "twisting" type motions of the HCH group. V5 and Y10 phi,psi bond rotations are also positively correlated, with their CbetaCalphaH groups undergoing similar "twisting" type motions. Motions of near-terminal residues G2 and G11 are less restricted and less correlated and are best described as wobbling-in-a-cone. V5 and Y10 side-chain motions, aside from being highly restricted, were found to be correlated with phi,psi bond rotations. At 303 K, where the hairpin is considered "unfolded," the peptide exists in a transient, collapsed state because backbone and side-chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG-based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.     

Propagated fluctuations of the electric potential in the apoplasm of Lepidium sativum L. roots

The electric potential on the surface of the Lepidium sativum L. root apex was recorded by means of six non-polarizable electrodes. Nonevoked fluctuations of the potential with amplitudes below 0.1 mV were observed. The fluctuations could be reversibly inhibited either by ether vapor or by anoxia caused by N₂. They did not occur in killed roots. Cross-correlation analysis of the fluctuations from six electrodes located one above another along the 3-mm apical region showed a pattern of time delay which indicates that the fluctuations may be the consequence of signals propagated in the root with a velocity of 3–9 mm · s^–1 in a basipetal direction from the root cap. We hypothesize that the fluctuations are due to signals of an unknown nature propagated along an intrasymplasmic continuous system, the symreticulum, composed of the cortical ER of individual cells and desmotubules passing through the plasmodesmata.Abbreviations AC alternating current - AP action potential - ACF autocorrelation function - CCF cross-correlation function - DC direct current - EEP extracellular electric potential This research was supported by Bundesminister für Forschung und Technologie, Bonn, and Ministerium für Wissenschaft und Forschung, Düsseldorf, (AGRAVIS). We are grateful to Mr. Dipl.-Ing. P. Blasczyk for constructing the amplifiers and for advice in instrumentation, and to Mr. H. Laubach for constructing the mechanical assembly.