Structure and dynamics of an unfolded protein examined by molecular dynamics simulation.

The accurate characterization of the structure and dynamics of proteins in disordered states is a difficult problem at the frontier of structural biology whose solution promises to further our understanding of protein folding and intrinsically disordered proteins. Molecular dynamics (MD) simulations have added considerably to our understanding of folded proteins, but the accuracy with which the force fields used in such simulations can describe disordered proteins is unclear. In this work, using a modern force field, we performed a 200 µs unrestrained MD simulation of the acid-unfolded state of an experimentally well-characterized protein, ACBP, to explore the extent to which state-of-the-art simulation can describe the structural and dynamical features of a disordered protein. By comparing the simulation results with the results of NMR experiments, we demonstrate that the simulation successfully captures important aspects of both the local and global structure. Our simulation was ~2 orders of magnitude longer than those in previous studies of unfolded proteins, a length sufficient to observe repeated formation and breaking of helical structure, which we found to occur on a multimicrosecond time scale. We observed one structural feature that formed but did not break during the simulation, highlighting the difficulty in sampling disordered states. Overall, however, our simulation results are in reasonable agreement with the experimental data, demonstrating that MD simulations can already be useful in describing disordered proteins. Finally, our direct calculation of certain NMR observables from the simulation provides new insight into the general relationship between structural features of disordered proteins and experimental NMR relaxation properties.

Crystal structures of beta-neurexin 1 and beta-neurexin 2 ectodomains and dynamics of splice insertion sequence 4.

Presynaptic neurexins (NRXs) bind to postsynaptic neuroligins (NLs) to form Ca(2+)-dependent complexes that bridge neural synapses. beta-NRXs bind NLs through their LNS domains, which contain a single site of alternative splicing (splice site 4) giving rise to two isoforms: +4 and Delta. We present crystal structures of the Delta isoforms of the LNS domains from beta-NRX1 and beta-NRX2, crystallized in the presence of Ca(2+) ions. The Ca(2+)-binding site is disordered in the beta-NRX2 structure, but the 1.7 A beta-NRX1 structure reveals a single Ca(2+) ion, approximately 12 A from the splice insertion site, with one coordinating ligand donated by a glutamic acid from an adjacent beta-NRX1 molecule. NMR studies of beta-NRX1+4 show that the insertion sequence is unstructured, and remains at least partially disordered in complex with NL. These results raise the possibility that beta-NRX insertion sequence 4 may function in roles independent of neuroligin binding.

Protein side-chain dynamics and residual conformational entropy.

Changes in residual conformational entropy of proteins can be significant components of the thermodynamics of folding and binding. Nuclear magnetic resonance (NMR) spin relaxation is the only experimental technique capable of probing local protein entropy, by inference from local internal conformational dynamics. To assess the validity of this approach, the picosecond-to-nanosecond dynamics of the arginine side-chain N(epsilon)-H(epsilon) bond vectors of Escherichia coli ribonuclease H (RNase H) were determined by NMR spin relaxation and compared to the mechanistic detail provided by molecular dynamics (MD) simulations. The results indicate that arginine N(epsilon) spin relaxation primarily reflects persistence of guanidinium salt bridges and correlates well with simulated side-chain conformational entropy. In particular cases, the simulations show that the aliphatic part of the arginine side chain can retain substantial disorder while the guanidinium group maintains its salt bridges; thus, the N(epsilon)-H(epsilon) bond-vector orientation is conserved and side-chain flexibility is concealed from N(epsilon) spin relaxation. The MD simulations and an analysis of a rotamer library suggest that dynamic decoupling of the terminal moiety from the remainder of the side chain occurs for all five amino acids with more than two side-chain dihedral angles (R, K, E, Q, and M). Dynamic decoupling thus may represent a general biophysical strategy for minimizing the entropic penalties of folding and binding.

Structural analysis of protein dynamics by MD simulations and NMR spin-relaxation.

Molecular dynamics (MD) simulations and nuclear magnetic resonance spin-relaxation measurements provide detailed insights into ps-ns structural dynamics of proteins. An analysis of discrepancies between the two methods is presented for the B3 immunoglobulin-binding domain of streptococcal protein G. MD simulations using three MD force fields (OPLS-AA, AMBER ff99SB, and AMBER ff03) overestimate the flexibility of backbone N--H vectors at the borders of secondary structure and in loops when compared with experimentally determined backbone amide generalized order parameters (Hall and Fushman, J Am Chem Soc 2006; 12:7855-7870). Comparison with a previous study of residual dipolar coupling constants (Bouvignies et al., Proc Natl Acad Sci USA 2005;102:13885-13890) indicates that slower timescale motions do not account for the discrepancies. Structural analysis reveals that relative imbalance between the description of hydrogen bonding and other terms of modern force fields may be responsible for disagreement.

Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins.

A molecular-level understanding of the function of a protein requires knowledge of both its structural and dynamic properties. NMR spectroscopy allows the measurement of generalized order parameters that provide an atomistic description of picosecond and nanosecond fluctuations in protein structure. Molecular dynamics (MD) simulation provides a complementary approach to the study of protein dynamics on similar time scales. Comparisons between NMR spectroscopy and MD simulations can be used to interpret experimental results and to improve the quality of simulation-related force fields and integration methods. However, apparent systematic discrepancies between order parameters extracted from simulations and experiments are common, particularly for elements of noncanonical secondary structure. In this paper, results from a 1.2 micros explicit solvent MD simulation of the protein ubiquitin are compared with previously determined backbone order parameters derived from NMR relaxation experiments [Tjandra, N.; Feller, S. E.; Pastor, R. W.; Bax, A. J. Am. Chem. Soc. 1995, 117, 12562-12566]. The simulation reveals fluctuations in three loop regions that occur on time scales comparable to or longer than that of the overall rotational diffusion of ubiquitin and whose effects would not be apparent in experimentally derived order parameters. A coupled analysis of internal and overall motion yields simulated order parameters substantially closer to the experimentally determined values than is the case for a conventional analysis of internal motion alone. Improved agreement between simulation and experiment also is encouraging from the viewpoint of assessing the accuracy of long MD simulations.

Using wavelet de-noised spectra in NMR screening.

Principal component analysis (PCA) is a commonly used algorithm in multivariate analysis of NMR screening data. PCA substantially reduces the complexity of data in which a large number of variables are interrelated. For series of NMR spectra obtained for ligand binding, it is commonly used to visually group spectra with a similar response to ligand binding. A series of filters are applied to the experimental data to obtain suitable descriptors for PCA which optimize computational efficiency and minimize the weight of small chemical shift variations. The most common filter is bucketing where adjacent points are summed to a bucket. To overcome some inherent disadvantages of the bucketing procedure we have explored the effect of wavelet de-noising on multivariate analysis, using a series of HSQC spectra of proteins with different ligands present. The combination of wavelet de-noising and PCA is most efficient when PCA is applied to wavelet coefficients. This new algorithm yields good clustering and can be applied to series of one- or two-dimensional spectra.

Double-quantum biased covariance spectroscopy: application to the 2D INADEQUATE experiment.

A novel processing scheme is presented that converts a two-dimensional double-quantum NMR spectrum into a single-quantum correlation spectrum. The covariance-like spectrum is computed from the 2D Fourier transform spectrum by emphasizing contributions that fulfill the double-quantum condition resulting in a symmetric spectrum that is easier to analyze. The method is demonstrated for the 2D INADEQUATE experiment.

Conformational preferences underlying reduced activity of a thermophilic ribonuclease H.

The conformational basis for reduced activity of the thermophilic ribonuclease HI enzyme from Thermus thermophilus, compared to its mesophilic homolog from Escherichia coli, is elucidated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. Explicit-solvent all-atom MD simulations of the two wild-type proteins and an E. coli mutant in which a glycine residue is inserted after position 80 to mimic the T. thermophilus protein reproduce the differences in conformational dynamics determined from (15)N spin-relaxation NMR spectroscopy of three loop regions that surround the active site and contain functionally important residues: the glycine-rich region, the handle region, and the ß5/αE loop. Examination of the MD trajectories indicates that the thermophilic protein samples conformations productive for substrate binding and activity less frequently than the mesophilic enzyme, although these differences may manifest as either increased or decreased relative flexibility of the different regions. Additional MD simulations indicate that mutations increasing activity of the T. thermophilus enzyme at mesophilic temperatures do so by reconfiguring the local environments of the mutated sites to more closely resemble active conformations. Taken together, the results show that both locally increased and decreased flexibility contribute to an overall reduction in activity of T. thermophilus ribonuclease H compared to its mesophilic E. coli homolog.

Covariance NMR spectroscopy by singular value decomposition.

Covariance NMR is demonstrated for homonuclear 2D NMR data collected using the hypercomplex and TPPI methods. Absorption mode 2D spectra are obtained by application of the square-root operation to the covariance matrices. The resulting spectra closely resemble the 2D Fourier transformation spectra, except that they are fully symmetric with the spectral resolution along both dimensions determined by the favorable resolution achievable along omega2. An efficient method is introduced for the calculation of the square root of the covariance spectrum by applying a singular value decomposition (SVD) directly to the mixed time-frequency domain data matrix. Applications are shown for 2D NOESY and 2QF-COSY data sets and computational benchmarks are given for data matrix dimensions typically encountered in practice. The SVD implementation makes covariance NMR amenable to routine applications.

Conformational Dynamics of the Partially Disordered Yeast Transcription Factor GCN4.

Molecular dynamics (MD) simulations have been employed to study the conformational dynamics of the partially disordered DNA binding basic leucine zipper domain of the yeast transcription factor GCN4. We demonstrate that back-calculated NMR chemical shifts and spin-relaxation data provide complementary probes of the structure and dynamics of disordered protein states and enable comparisons of the accuracy of multiple MD trajectories. In particular, back-calculated chemical shifts provide a sensitive probe of the populations of residual secondary structure elements and helix capping interactions, while spin-relaxation calculations are sensitive to a combination of dynamic and structural factors. Back calculated chemical shift and spin-relaxation data can be used to evaluate the populations of specific interactions in disordered states and identify regions of the phase space that are inconsistent with experimental measurements. The structural interactions that favor and disfavor helical conformations in the disordered basic region of the GCN4 bZip domain were analyzed in order to assess the implications of the structure and dynamics of the apo form for the DNA binding mechanism. The structural couplings observed in these experimentally validated simulations are consistent with a mechanism where the binding of a preformed helical interface would induce folding in the remainder of the protein, supporting a hybrid conformational selection / induced folding binding mechanism.

Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins.

Despite major technical advance in methods used for structural investigations of proteins structure determination of membrane proteins still poses a significant challenge. Recently, the application of cell-free expression systems to membrane proteins has demonstrated that this technique can be used to produce quantities sufficient for structural investigations for many different membrane proteins. In particular for NMR spectroscopy, cell-free expression provides major advantages since it allows for amino acid type selective and even amino acid position specific labeling. In this mini-review we discuss the combination of cell-free membrane protein expression and liquid state NMR spectroscopy.

Efficient strategy for the rapid backbone assignment of membrane proteins.

Investigations of membrane proteins pose one of the biggest current challenges in structural biology. Recent advances in protein production techniques based on cell-free transcription/translation methods have, however, opened new opportunities in this area. Here, we report an efficient protocol for the backbone assignment of membrane proteins as the first step of NMR-based structure determination.