Dynamics Of Proteins In Solution Research Articles

Chirality Transfer Effects in Proline-Substituted Coumarin Compounds

Conformations of proline-substituted chromophores are determined by using circular dichroism (CD) spectroscopy and quantum chemistry calculation method. Coumarin is chosen for the optical chromophore and proline amino acid is attached to its C7 position. The coumarin-proline conjugate considered contains both fluorophore and peptide linker where any polypeptides or biomolecules can be additionally connected to the free carboxyl group of the proline. Thus, the coumarin-proline is a potentially useful composite chirality-probe system for studies of protein dynamics in solution. However, detailed conformation of coumarin ring with respect to the proline ring has to be determined first. We found that there are two possible conformers, which differ from each other by the relative orientation of the coumarin ring. Comparing the measured CD spectra with the calculated ones, we directly show that only one of the two conformers is dominant in polar solvents except for water. The present study suggests that the local structure around an optical chromophore, when it is introduced to polypeptides or other biomolecules, can be studied by examining the electronic optical activity of the probe chromophore, as long as the chirality transfer from the attached amino acid to the chromophore is significantly large.

Protein Hydrogen Exchange Measured at Single-Residue Resolution by Electron Transfer Dissociation Mass Spectrometry

Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation. The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas-phase hydrogen (1H/2H) migration (i.e., hydrogen scrambling). This article demonstrates that ETD can be implemented in a mass spectrometric method to monitor the conformational dynamics of proteins in solution at single-residue resolution.

Calculations of intermode coupling constants and simulations of amide I, II, and III vibrational spectra of dipeptides

Amide I, II, and III vibrations of polypeptides are important marker modes whose vibrational spectra can provide critical information on structure and dynamics of proteins in solution. The extent of delocalization and vibrational properties of amide normal mode can be described by the amide local mode frequencies and intermode coupling constants between a pair of amide local modes. To determine these fundamental quantities, the previous Hessian matrix reconstruction method has been generalized here and applied to the density functional theory results for various dipeptide conformers. The calculation results are then used to simulate IR absorption, vibrational circular dichroism, and 2D IR spectra of dipeptides. The relationships between dipeptide backbone conformations and these vibrational spectra are discussed. It is believed that the present computational method and results will be of use to quantitatively simulate vibrational spectra of complicated polypeptides beyond simple dipeptides

Electron Transfer Dissociation Facilitates the Measurement of Deuterium Incorporation into Selectively Labeled Peptides with Single Residue Resolution

Mass spectrometry is routinely applied to measure the incorporation of deuterium into proteins and peptides. The exchange of labile, heteroatom-bound hydrogens is mainly used to probe the structural dynamics of proteins in solution, e.g., by hydrogen-exchange mass spectrometry, but also to study the gas-phase structure and fragmentation mechanisms of polypeptide ions. Despite considerable effort in recent years, there is no widely established mass spectrometric method to localize the incorporated deuterium to single amino acid residues, and typically, only the overall deuterium content of peptides or proteins is obtained. The main reason for this is that CID and related techniques induce intramolecular migration of hydrogens ("hydrogen scrambling") upon vibrational excitation of the even-electron precursor ion, thus randomizing the positional distribution of the incorporated deuterium atoms before fragmentation. In contrast, decomposition of radical gas-phase peptide cations upon electron capture dissociation was recently demonstrated to proceed with a very low level of amide hydrogen scrambling. Employing model peptides developed to enable sensitive detection of hydrogen scrambling, we show in the present study that electron transfer dissociation in a 3D-quadrupole ion trap retains the site-specific solution-phase deuterium incorporation pattern and allows for localization of incorporated deuterium with single residue resolution. Furthermore, we exploit this finding to monitor how collisional activation induces proton mobility in a gaseous peptide ion at various levels of vibrational excitation.

Erratum: Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

Nat. Methods 5, 881–886 (2008); published online 21 September 2008; corrected after print 29 September 2008. In the version of this article initially published, the time scale reported in the Figure 2d legend is incorrect. The correct time scale should be 3 μs. Additionally, the time delay of 320 msreported in Figure 5b is incorrect.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

Effects of electrospray capillary temperature on amide hydrogen exchange

Amide hydrogen/deuterium (H/D) exchange coupled with proteolysis, high-perfeomance liquid chromatographic (HPLC) separation and mass spectrometry (MS) has become a powerful tool to study protein dynamics in solution. Prior to the execution of H/D exchange experiments, various experimental parameters have to be set, including proteolysis, HPLC, and MS conditions. Here we investigate the effects of electrospray capillary temperature on deuterium retention in backbone amides of various pepsin-generated cytochrome c peptides. Lower capillary temperature generally helps retain more deuterium than higher capillary temperature. When the capillary temperature was 150 degrees C, on average 26% more deuterium was retained than when the capillary temperature was set at 250 degrees C. The effects of capillary temperature varied depending on the ions monitored. There was little difference in deuterium retention among different charge state species of the same peptide at 150 degrees C. However, a lower charge state ion loses more deuterium atoms going from 150 degrees C to 250 degrees C than the corresponding higher charge state species. These results indicate that the capillary temperature should be optimized not only to maximize the signal-to-noise of each ion followed in H/D exchange experiments, but also to minimize the deuterium loss of the ions. Also the loss of deuterium in several ions, especially lower charge state ones, should be monitored in the optimization, as the temperature effects vary among ions and are more significant for lower charge state ions.

Towards the prediction of NMR relaxation rates in proteins from their structure by a network of coupled rotators

Abstract During the past decades, NMR spectroscopy of isotopically labelled proteins has emerged as a unique tool for the study of internal protein dynamics in solution. The possibility of measuring spin relaxation rates in proteins has motivated numerous theoretical and methodological developments aiming at the interpretation and the prediction of their internal dynamics. In this article, we discuss the possibility of predicting 15N relaxation rates using a Network of Coupled Rotators to describe internal motions of a protein starting from its three-dimensional structure, and illustrate the approach by the example of the protein calbindin.

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

S3d2-1 The use of the third generation synchrotron small-angle scattering on protein dynamics in solution : present and future(S3-d2: "Structural approach to protein dynamics using solution scattering",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S3d2-1 The use of the third generation synchrotron small-angle scattering on protein dynamics in solution : present and future(S3-d2: "Structural approach to protein dynamics using solution scattering",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

A New Spin Probe of Protein Dynamics: Nitrogen Relaxation in15N−2H Amide Groups

(15)N spin relaxation data have provided a wealth of information on protein dynamics in solution. Standard R(1), R(1)(rho), and NOE experiments aimed at (15)N[(1)H] amide moieties are complemented in this work by HA(CACO)N-type experiments allowing the measurement of nitrogen R(1) and R(1)(rho) rates at deuterated (15)N[(2)D] sites. Difference rates obtained using this approach, R(1)((15)N[(1)H]) - R(1)((15)N[(2)D]) and R(2)((15)N[(1)H]) - R(2)((15)N[(2)D]), depend exclusively on dipolar interactions and are insensitive to (15)N CSA and R(ex) relaxation mechanisms. The methodology has been tested on a sample of peptostreptococcal protein L (63 residues) prepared in 50% H(2)O-50% D(2)O solvent. The results from the new and conventional experiments are found to be consistent, with respect to both local backbone dynamics and overall protein tumbling. Combining several data sets permits evaluation of the spectral density J(omega(D) + omega(N)) for each amide site. This spectral density samples a uniquely low frequency (26 MHz at a 500 MHz field) and, therefore, is expected to be highly useful for characterizing nanosecond time scale local motions. The spectral density mapping demonstrates that, in the case of protein L, J(omega(D) + omega(N)) values are compatible with the Lipari-Szabo interpretation of backbone dynamics based on the conventional (15)N relaxation data.

Molecular structure and dynamics of proteins in solution: Insights derived from high-resolution NMR approaches

This article reviews heteronuclear NMR techniques that are used to characterize the 3D structures and internal molecular dynamics of proteins in solution. Applications to several proteins that have been studied in our laboratory are presented. Topics discussed include sequential assignments, the use of nuclear Overhauser enhancement spectroscopy (NOESY) and residual dipolar coupling restraints to determine protein structure, and the use of relaxation measurements to obtain information about protein internal motions. Also discussed are recent computational approaches directed at increasing the efficiency of structure determination, as well as new methods for studying large proteins.

Editorial: Fluorescence spectroscopy and nucleic acids

Fluorescence spectroscopy is an established tool for investigating the structure, folding, and conformational dynamics of proteins in solution. The widespread use of fluorescence techniques for the physicochemical characterization of proteins stems from the occurrence of the naturally fluorescent amino acids tryptophan and tyrosine in many proteins and the great sensitivity of the emission properties of these amino acids to perturbations in protein structure. Historically, the application of fluorescence spectroscopy to nucleic acids has been hindered by the lack of suitable intrinsic fluorophores (the exception are certain tRNAs that contain unusual fluorescent bases). However, with the advent of rapid and efficient methods for the solid phase synthesis and site-specific fluorescent labeling of DNA and RNA oligonucleotides, the power of fluorescence spectroscopy can now be brought to bear on nucleic acids. Indeed, the past decade has witnessed a tremendous growth in the number of studies in which fluorescence spectroscopic techniques have been used to characterize the structural properties and fundamental biophysical behaviors of nucleic acids and nucleic acid–protein complexes. Particular advantages of fluorescence-based methods include high detection sensitivity, fast time resolution, and ready adaptability to a wide range of solution conditions. Of the many spectroscopic phenomena that can be exploited to examine nucleic acids, fluorescence resonance energy transfer (FRET) lends itself to the broadest range of applications. This issue of Nucleic Acid Sciences outlines some of the methodologies employed and illustrates the rich variety of information that is forthcoming from FRET-based analyses of nucleic acids, highlighting studies that focus on structural characterization, thermodynamic properties, and kinetic processes. Since the efficiency of resonance energy transfer from a donor fluorophore to an acceptor reports on distances ranging from 10 to 100 Å, measurements of FRET can be used to define the global structure of nucleic acid complexes. This approach is especially powerful in the case of large or disordered complexes that are not amenable to structural analysis by x-ray crystallographic or NMR spectroscopic methods. Showing the broad applicability of this technique to structural questions, FRET has been used to elucidate the helical geometry of multistranded nucleic acid complexes (Singleton and Xiao), to explore the dynamic solution structure of branched DNA molecules (Klostermeier and Millar), to characterize the conformation of bent DNA (Parkhurst et al.), and to probe the global architecture of large macromolecule complexes (Heyduk and Niedziela-Majka). In addition to its utility in probing structural questions, FRET may be used to analyze nucleic acid complex stability and conformational transitions. Precise thermodynamic characterization of the impact of base lesions on DNA duplex stability is possible using sensitive FRET-based methods, giving insight into the biological consequences of DNA damage and the molecular basis of mutagenesis (Plum and Breslauer). As another example, the time-resolved FRET technique can be used to identify and quantify different tertiary structure conformers of RNA, which often play a role in regulating biological activity. This approach has been used to investigate the molecular forces that drive conformational transitions in the hairpin ribozyme (Klostermeier and Millar). Finally, FRET methods can also be harnessed to study the kinetics of nucleic acid conformational transitions, underscoring the great versatility of fluorescence techniques for biophysical characterization of nucleic acids. FRET and rapid kinetic methods have been used to study the dynamics of DNA bending by the transcription factor TBP in real time and to establish a detailed kinetic mechanism for the interaction of TBP with DNA (Parkhurst et al.). Similar methods have been used to monitor large-scale conformational changes during the catalytic cycle of the hairpin and hepatitis delta virus ribozymes (Walter et al.). The studies reviewed in this issue of Nucleic Acid Sciences illustrate the ability of fluorescence techniques to provide structural, thermodynamic, and kinetic information on a variety of biologically important DNA and RNA molecules. It is hoped that these examples will stimulate an even wider application of fluorescence techniques in the study of nucleic acids. While most of the examples presented here involve relatively short oligonucleotides, it is anticipated that the different fluorescent labeling strategies discussed herein will be combined to construct much larger DNA or RNA molecules. Thus, the fluorescence spectroscopic techniques described in this issue can be applied to a variety of dynamic macromolecular complexes, including the replisome, transcription complexes, and the ribosome. In addition, the fluorescence techniques can be extended to the single-molecule level, which should provide new insights into the conformational dynamics and biological function of nucleic acids (single-molecule fluorescence studies have been reviewed recently; T. J. Ha, Current Opinion in Structural Biology, 2001 Vol. 11, pp. 287–292).

Comparison of the Time‐resolved Absorption and Phosphorescence from the Tryptophan Triplet State in Proteins in Solution

Measurement of the room temperature Trp triplet state lifetime in proteins by time-resolved phosphorescence can provide valuable information on the structure and dynamics of proteins in solution. Our time-resolved absorption measurements on the long-lived states resulting from electronic excitation of the chromophore demonstrate the presence of more complex behavior than revealed by time-resolved phosphorescence. To provide additional insight into this behavior, a comparative study of time-resolved transient absorption and time-resolved phosphorescence of proteins in solution was carried out. The results show that the time evolution of the long-lived states observed through transient absorption often differs considerably from that observed in time-resolved phosphorescence. In some proteins, the presence of competing reactions complicates the interpretation of the transient absorption measurements (which may affect the phosphorescence yield). A more complete characterization of these processes will likely prove useful in the study of protein structure and dynamics in solution.

Study of protein dynamics in solution by measurement of 13C?-13CO NOE and 13CO longitudinal relaxation

(13)C(α)-(13)CO homonuclear NOE and (13)CO T(1) relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The (13)C(α)-(13)CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.

Comparison of the three-dimensional molecular models of bovine submicellar caseins with small-angle X-ray scattering. Influence of protein hydration

To test the applicability of two energy-minimized, three-dimensional structures of the bovine casein submicelle, theoretical small-angle X-ray scattering curves in the presence and absence of water were compared to experimental data. The published method simulates molecular dynamics of proteins in solution by employing adjustable Debye-Waller temperature factors (B factors) for the protein, for the solvent, and for protein-bound water. The programs were first tested upon bovine pancreatic trypsin inhibitor beginning with its known X-ray crystal structure. To approximate the degree of protein hydration previously determined by NMR relaxation experiments (0.01 g water/g protein), 120 water molecules were docked into the large void of the kappa-casein portion of the structure for both the symmetric and asymmetric casein submicelle models. To approximate hydrodynamic hydration (0.244 g water/g protein), 2703 water molecules were added to each of the above structures using the "droplet" algorithm in the Sybyl molecular modeling package. All structures were then energy-minimized and their solvation energies calculated. Theoretical small-angle X-ray scattering curves were calculated for all unhydrated and hydrated structures and compared with experimentally determined scattering profiles for submicellar casein. Best results were achieved with the 120-bound-water structure for both the symmetric and asymmetric submicelle models. Comparison of results for the protein submicelle models with those for the theoretical and literature values of bovine pancreatic trypsin inhibitor demonstrates the applicability of the methodology.

Overall and internal protein dynamics in solution studied by the nonselective proton relaxation.

A new algorithm for the analysis of nonselective proton relaxation data in protein solution is presented. T1 and T2 of protein protons in lysozyme and RNase solutions were measured at three resonance frequencies--11, 27 and 90 MHz. In addition we measured water T1 dispersions in lysozyme solutions over the frequency range of 10 kHz--10 MHz on a field-cycling installation. It was found that the correlation function of protein Brownian tumbling as a whole is nonexponential: in addition to a component with the usual correlation time tau t it contained also a component with a correlation time exceeding tau t by approximately an order of magnitude and with a small relative amplitude. The experiment shows that the parameters of the slow component of the tumbling correlation function depend both on the concentration and on the pH of the protein solution. To explain the results obtained one must take into account the interprotein electrostatic interactions in solution. All protein molecules in solution experience electrostatic torques from their neighbors and this gives rise to an anisotropy in the protein Brownian tumbling. The lifetime of this anisotropy is controlled by the translational diffusion of proteins.

Atomic solvation parameters applied to molecular dynamics of proteins in solution

A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor-to-water free energies of transfer of amino acid side-chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., & Karplus, M., 1983, J. Comput. Chem. 4(2), 187-217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26-residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231-244; Lauterwein, J., Brown, L.R., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219-230).

Accurate simulation of protein dynamics in solution.

Simulation of the molecular dynamics of a small protein, bovine pancreatic trypsin inhibitor, was found to be more realistic when water molecules were included than when in vacuo: the time-averaged structure was much more like that observed in high-resolution x-ray studies, the amplitudes of atomic vibration in solution were smaller, and fewer incorrect hydrogen bonds were formed. Our approach, which provides a sound basis for reliable simulation of diverse properties of biological macromolecules in solution, uses atom-centered forces and classical mechanics.

Conformations and conformational dynamics of proteins in solution studied by nuclear magnetic double resonance

Saturation of single resonances in the proton nuclear magnetic resonance (NMR) spectrum of a protein in solution results in changes in intensities of other resonances. These changes come about either through nuclear Overhauser effects which arise because of dipolar coupling between the protons in the molecule, or through cross-saturation effects which arise when the protein fluctuates between different well defined conformations. For lysozyme from hen egg white (mol. wt. approx. 14,500) the method reveals details of the protein structure and of individual atom fluctuations.