Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13Cα nuclear spin relaxation

Current methods of determining the rotational diffusion tensors of proteins in solution byNMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins.However, the distributions of orientations of N-H bond vectors are not isotropic in manyproteins, and correlations between bond vector orientations reduce the accuracy and precisionof rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both13C and 15N spin relaxation rate constants increases the robustness of the diffusiontensor analysis because the orientations of the C-H bond vectors differ from theorientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k,granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributionsof N-H and C-H bond vectors, are used to illustrate the advantages of thesimultaneous utilization of 13C and 15N relaxation data.     

The use of TROSY for detection and suppression of conformational exchange NMR line broadening in biological macromolecules

The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled ¹H and ¹⁵N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [¹⁵N,¹H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base ¹H–¹⁵N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D ^h1 J _HN,^h2 J _NN-quantitative [¹⁵N,¹H]-TROSY to measure ^h1 J _HN and ^h2 J _NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (^h2 J _NN+^h1 J _NH)-correlation-[¹⁵N,¹H]-TROSY to correlate ¹H^N chemical shifts of bases with the chemical shifts of the tertiary ¹⁵N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids.     

Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.     

Off-resonance effects in 15N T2 CPMG measurements

The systematic difference between T ₂ values obtained from CPMG and T ₁ experiments was observed for backbone ¹⁵N nuclei of bacterial ribonuclease barnase. Theoretical consideration suggests that the observed difference is caused by off-resonance effects of 180° pulses of the CPMG pulse train. Namely, at off-resonance conditions T ₁-dependent secondary echo coherence pathways considerably contribute to the signal decay in the CPMG experiment and result in systematic (up to 10%) offset-dependent overestimation of ¹⁵N T ₂ measured by the CPMG technique. Under certain circumstances off-resonance effects result in dependence of ¹⁵N T ₂ on CPMG frequency, which might be erroneously interpreted as conformational exchange on the millisecond time-scale. A procedure for numerical correction of ¹⁵N T ₂ (CPMG) data is proposed.     

The design and use of a hydraulically operated upward pressure-jump

The construction of hydraulically operated upward pressure-jump is described. Previous instruments with a comparable rate of pressration used helium gas to pressurize an observation cell to approximately half of the operating pressure reached with the present design. The above instrument, which can pressurize the observation cell in 1.8–3.8 ms. is safe to use and has a maximum working pressure of 30 MPa (300 atm). The device is compact in design and can easily be fitted to any observation cell capable of withstanding the transient change in pressure and of effectivekt damping cell resonances. The instrument finds specific application in the study of macromolecular equilibria with a large difference in volume between the equilibrium partners. This allow the system to be perturbed far from the equilibrium position at atmospheric pressure; the relaxations obtained can be analysed as essentially unidirectional processes. Kinetic data for the reaction would be obtained from experiments in a conventional downward pressure-jump. A study on the kinetics of the self-assembly of the myosin thick-filament is presented as a practical example.     

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R_1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide ¹H off-resonance R_1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using ¹⁵N or ¹³C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The ¹H off-resonance R_1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for ¹⁵N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly ¹⁵N-enriched samples that were either highly enriched in ²H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the ¹H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present ¹H off-resonance R_1ρ experiments are in excellent agreement with those determined previously using a combination of ¹⁵N laboratory-frame and off-resonance R_1ρ relaxation data, with average values of and 21 ± 3 μs, respectively.     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

Kinetics of pigment-acceptor interaction induced by continuous illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and light

The kinetics of dark reduction of chlorophyll P700 oxidized by continuous light in preparations of photosystem I reaction centers from cyanobacterium Synechosystis spharoides cooled in the dark to 160 K is essentially nonexponential. The characteristic times of the components range from fractions of a second to minutes or more. During the cooling of reaction center preparations under illumination with actinic light, most of the chlorophyll P700 molecules are fixed in the oxidized state at 160 K. The kinetics of dark reduction of P700⁺ in the fraction of reaction centers that retain photochemical activity under these conditions is somewhat faster compared to the samples cooled in the dark. A theoretical analysis of substantial deceleration of P700⁺ dark recovery kinetics was done for preparations of photosystem I reaction centers oxidized by continuous light at 160 K in comparison to the experiments where reaction centers were oxidized by short single light flashes. This slowing down of the kinetics in samples excited by continuous illumination can be explained by microconformational relaxation processes related to proton shifts in the reaction center.     

Assessment of the validity of the Adams and Fujita approximation for the higher oligomers of human spectrin

Analysing the self-association behaviour of human erythrocyte spectrin is complicated by a large degree of nonideality. Adams and Fujita [1] proposed that, as a first order approximation, the logarithm of the activity coefficient of the protomer of a self-associating system can be considered to be linearly dependent on the total concentration of the protein, and that the same second virial coefficient could be considered to apply to all species. As a consequence of the Adams and Fujita approximation, the apparent equilibrium constant is equal to the thermodynamic equilibrium constant. The equilibrium concentrations at 30°C of each oligomer spectrin species up to the 14-mer were determined after electrophoresis at low temperature. An apparent equilibrium constant for forming tetramer (K_2,4) of (1.2 ± 0.1) × 10⁶ l/mol was obtained, a value of (9.4 ± 0.7) × 10⁴ l/mol was obtained for K_4,6 and for all reactions forming oligomers higher than the hexamer an average approximate value of (2.7 ± 0.4) × 10⁵ l/mol was obtained. The apparent equilibrium constants for the formation of all oligomer species of spectrin up to the tetrakaidecamer (14-mer) remain relatively independent of total spectrin concentration, and indicate that within the precision of the measurements a single virial coefficient is sufficient to account for the nonideality of spectrin self-association over the range 2–42 g/l, thus further justifying the use of the Adams and Fujita approximation for this protein over this concentration range.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin

The N-terminal region of non-erythroid alpha spectrin (SpαII) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with SpαII. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in SpαII, V22D, which interferes with the coiled coil bundling of SpαII with beta spectrin, also affects SpαII interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with SpαII. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.     

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium

In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts (`conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2I_xS_x and 2I_yS_y, induced by the differential relaxation. The effect is illustrated with an application to ¹³C,¹⁵N-labeled quail CRP2(LIM2), by studying ¹⁵N-¹H^N multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two -strands in CRP2(LIM2). Differential ¹H^N and ¹⁵N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins.     

Self-Organization in Living Cells: Networks of Protein Machines and Nonequilibrium Soft Matter

Microscopic self-organization phenomena inside a living cell should not represent merely a reduced copy of self-organization in macroscopic systems. A cell is populated by active protein machines that communicate via small molecules diffusing through the cytoplasm. Mutual synchronization of machine cycles can spontaneously develop in such networks – an effect which is similar to coherent laser generation. On the other hand, an interplay between reactions, diffusion and phase transitions in biological soft matter may lead to the formation of stationary or traveling nonequilibrium nanoscale structures.