Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed.     

Solvent interactions and protein dynamics in spin-labeled T4 lysozyme

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a "hinge" residue, which explains the high degree of flexibility and motional freedom at this site.     

Comparison of backbone dynamics of oxidized and reduced putidaredoxin by 15N NMR relaxation measurements.

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

Characterization of the formation of amyloid protofibrils from barstar by mapping residue-specific fluorescence dynamics

The small protein barstar aggregates at low pH to form soluble oligomers, which can be transformed into fibrillar aggregates at an elevated temperature. To characterize structurally, with residue-specific resolution, the process of amyloid formation of barstar, as well as to monitor the increase in size that accompanies the aggregation process, time-resolved fluorescence anisotropy decay measurements have been introduced as a valuable probe. Seven different single-cysteine-containing mutant forms of barstar were made, to each of which a fluorophore was attached at the thiol group. The rotational dynamics of these seven fluorophores, as well as of the sole intrinsic tryptophan residue in the protein, were determined in the amyloid protofibrils formed, as well as in the soluble oligomers from which the protofibrils arise upon heating. Mapping of the fast rotational dynamics onto the sequence of the protein yields dynamic amplitude maps that allowed identification of the segments of the chain that possess local structure in the soluble oligomer and amyloid protofibrils. The patterns of these maps of the soluble oligomer and protofibrils are seen to be similar; and protofibrils display more local structure than do the soluble oligomers, at all residue positions studied. The observation that transformation from soluble oligomers to protofibrils does not perturb local structure significantly at eight different residue positions, suggests that the soluble oligomers transform directly into protofibrils, without undergoing drastic structural rearrangements.     

Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain

The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of alpha-helices and beta-sheets, with a marginal change in beta-sheet content occurring upon phosphorylation.     

Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study

100-ns molecular dynamics simulations of fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, both pure and containing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl-chain labeled fluorescent analogs (C6-NBD-PC and C12-NBD-PC), are described. These molecules are widely used as probes for lipid structure and dynamics. The results obtained here for pure DPPC agree with both experimental and theoretical published works. We verified that the NBD fluorophore of both derivatives loops to a transverse location closer to the interface than to the center of the bilayer. Whereas this was observed previously in experimental literature works, conflicting transverse locations were proposed for the NBD group. According to our results, the maximum of the transverse distribution of NBD is located around the glycerol backbone/carbonyl region, and the nitro group is the most external part of the fluorophore. Hydrogen bonds from the NH group of NBD (mostly to glycerol backbone lipid O atoms) and to the nitro O atoms of NBD (from water OH groups) are continuously observed. Rotation of NBD occurs with approximately 2.5-5 ns average correlation time for these probes, but very fast, unresolved reorientation motions occur in <20 ps, in agreement with time-resolved fluorescence anisotropy measurements. Finally, within the uncertainty of the analysis, both probes show lateral diffusion dynamics identical to DPPC.     

Solvent density and long-range dipole field around a DNA-binding protein studied by molecular dynamics

The distribution and orientation of solvent around a DNA-binding protein, 434 Cro, were investigated by molecular dynamics simulations with a periodic-boundary condition. The protein was treated in two states: charged and neutral. The computed high-density sites of the solvent around the protein correlated well with the experimentally determined crystal-water sites, in both the charged and neutral states. A local density map, introduced to investigate the solvent density around the highly mobile regions of the protein, showed a hydration shell around hydrophobic sidechains and hydrogen-bondable sites around hydrophilic sidechains, and also showed that the solvent density is sensitive to the slight concaves of the sidechain surface. The long-range solvent-dipole field was observed around the protein, where the pattern of the dipole ordering was considerably different between the charged and neutral states. A local solvent-dipole field was introduced, and the pattern of the dipole ordering was different between the hydrophobic and hydrophilic sidechains. The dipole field from the charged state provided a higher correlation to the electrostatic field obtained from the Poisson-Boltzmann's equation than that from the neutral state, although the correlation become weak quickly for the both states with increasing the protein-solvent distance.     

Solution structure and dynamics of the functional domain of Paracoccus denitrificans cytochrome c(552) in both redox states

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.     

Stability and dynamics of domain-swapped bovine-seminal ribonuclease

The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5 ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks.     

Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study

100-ns molecular dynamics simulations of fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, both pure and containing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl-chain labeled fluorescent analogs (C6-NBD-PC and C12-NBD-PC), are described. These molecules are widely used as probes for lipid structure and dynamics. The results obtained here for pure DPPC agree with both experimental and theoretical published works. We verified that the NBD fluorophore of both derivatives loops to a transverse location closer to the interface than to the center of the bilayer. Whereas this was observed previously in experimental literature works, conflicting transverse locations were proposed for the NBD group. According to our results, the maximum of the transverse distribution of NBD is located around the glycerol backbone/carbonyl region, and the nitro group is the most external part of the fluorophore. Hydrogen bonds from the NH group of NBD (mostly to glycerol backbone lipid O atoms) and to the nitro O atoms of NBD (from water OH groups) are continuously observed. Rotation of NBD occurs with ∼ 2.5-5 ns average correlation time for these probes, but very fast, unresolved reorientation motions occur in < 20 ps, in agreement with time-resolved fluorescence anisotropy measurements. Finally, within the uncertainty of the analysis, both probes show lateral diffusion dynamics identical to DPPC.     

Probing the molecular dynamics of the ABC multidrug transporter LmrA by deuterium solid-state nuclear magnetic resonance

The molecular dynamics of the 64 kDa ABC multidrug efflux pump LmrA from Lactococcus lactis within lipid membranes has been investigated by deuterium solid-state NMR. Deuteriomethyl-labeled alanine has been used to probe global protein backbone dynamics. A comparison of static deuterium NMR spectra of full-length LmrA in the resting state and its isolated transmembrane domain revealed a high mobility for the nucleotide binding domains. Their motional freedom is restricted upon ATP binding as seen for LmrA in complex with AMP-PNP, a nonhydrolyzable ATP analogue. LmrA returns to full motional flexibility in the posthydrolysis, vanadate-trapped state. These experiments provide insight into the molecular dynamics of a full-length ABC transporter during the catalytic cycle. Data are discussed in the context of known biochemical data and structural models of ABC transporters.     

Molecular dynamics of sugars bound to wheat germ agglutinin, as studied by deuterium nuclear magnetic resonance.

Deuterium nuclear magnetic resonance is used to delineate the molecular dynamics of sugars bound to a lectin. 2H spin-spin relaxation times (from linewidth measurements) and reorientational correlation times are determined for N-acetylglucosamine specifically-labeled with 2H in the N-acetyl group and at carbon-3 of the pyranoside ring, in the presence and absence of wheat germ agglutinin. The correlation time for the 2H-label of N-acetylglucosamine-3-2H in the bound state is the same as that of the protein (3 X 10(-8)S), indicating that the six-membered ring has negligible motional freedom relative to the protein. The correlation time for the C2H3 group of N-acetyl-2H3-glucosamine (1.7 X 10(-9)S) shows that the N-acetyl side chain is also immobilized in the binding site, the only motion available being rotation of the C2H3 group about its threefold axis.     

Protein dynamics in supercooled water: the search for slow motional modes

The impact of studying protein dynamics in supercooled water for identifying slow motional modes on the s time scale is demonstrated. Backbone ¹⁵N spin relaxation parameters were measured at –13°C for ubiquitin, which plays a central role for signaling proteolysis, cellular trafficking and kinase activation in eukaryotic organisms. A hitherto undetected motional mode involving Val 70 was found, which may well play an important role for ubiquitin recognition. The measurement of rotating frame ¹⁵N relaxation times as a function of the spin-lock field allowed determination of the correlation time of this motional mode, which would not have been feasible above 0°C.     

DNA dynamics in RecA-DNA filaments: ATP hydrolysis-related flexibility in DNA

RecA-catalyzed DNA recombination is initiated by a mandatory, high-energy form of DNA in RecA-nucleoprotein filaments, where bases are highly unstacked and the backbone is highly unwound. Interestingly, only the energetics consequent to adenosine triphosphate (ATP) binding, rather than its hydrolysis, seems sufficient to mediate such a high-energy structural hallmark of a recombination filament. The structural consequence of ATP hydrolysis on the DNA part of the filament thus remains largely unknown. We report time-resolved fluorescence dynamics of bases in RecA-DNA complexes and demonstrate that DNA bases in the same exhibit novel, motional dynamics with a rotational correlation time of 7-10 ns, specifically in the presence of ATP hydrolysis. When the ongoing ATP hydrolysis of RecA-DNA filament is "poisoned" by a nonhydrolyzable form of ATP (ATPgammaS), the motional dynamics cease and reveal a global motion with a rotational correlation time of >20 ns. Such ATP hydrolysis-induced flexibility ensues in single-stranded as well as double-stranded bases of RecA-DNA filaments. These results suggest that the role of ATP hydrolysis is to induce a high level of backbone flexibility in RecA-DNA filament, a dynamic property that is likely to be important for efficient strand exchanges in ATP hydrolysis specific RecA reactions. It is the absence of these motions that may cause high rigidity in RecA-DNA filaments in ATPgammaS. Dynamic light scattering measurement comparisons of RecA-ss-DNA filaments formed in ATPgammaS vs that of ATP confirmed such an interpretation, where the former showed a complex of larger (30 nm) hydrodynamic radius than that of latter (12-15 nm). Taken together, these results reveal a more dynamic state of DNA in RecA-DNA filament that is hydrolyzing ATP, which encourage us to model the role of ATP hydrolysis in RecA-mediated DNA transactions.     

Solvent Interactions and Protein Dynamics in Spin-labeled T4 Lysozyme

Abstract

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a “hinge” residue, which explains the high degree of flexibility and motional freedom at this site.     

Backbone dynamics in an intramolecular prolylpeptide-SH3 complex from the diphtheria toxin repressor, DtxR

The diphtheria toxin repressor contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment and stabilizes the inactive state of the repressor. Upon activation of diphtheria toxin repressor (DtxR) by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. Here we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined using NMR spectroscopy and molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times, while residues in the loops connecting β-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment exhibited low-order parameters with internal rotational correlation times on the order of 0.6 ns-1 ns. Further analysis showed that the SH3 domain was rich in millisecond time scale motions while the Pr segment exhibited motions on the 100 μs time scale. Molecular dynamics simulations indicated structural rearrangements that may contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The results here provide new insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.     

Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T?cell Receptor Binding Loops

Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops.