Using NMR diffusion data to validate MD models of disordered proteins: Test case of N-terminal tail of histone H4.

MD simulations can provide uniquely detailed models of intrinsically disordered proteins (IDPs). However, these models need careful experimental validation. The coefficient of translational diffusion Dtr, measurable by pulsed field gradient NMR, offers a potentially useful piece of experimental information related to the compactness of the IDP's conformational ensemble. Here, we investigate, both experimentally and via the MD modeling, the translational diffusion of a 25-residue N-terminal fragment from histone H4 (N-H4). We found that the predicted values of Dtr, as obtained from mean-square displacement of the peptide in the MD simulations, are largely determined by the viscosity of the MD water (which has been reinvestigated as a part of our study). Beyond that, our analysis of the diffusion data indicates that MD simulations of N-H4 in the TIP4P-Ew water give rise to an overly compact conformational ensemble for this peptide. In contrast, TIP4P-D and OPC simulations produce the ensembles that are consistent with the experimental Dtr result. These observations are supported by the analyses of the 15N spin relaxation rates. We also tested a number of empirical methods to predict Dtr based on IDP's coordinates extracted from the MD snapshots. In particular, we show that the popular approach involving the program HYDROPRO can produce misleading results. This happens because HYDROPRO is not intended to predict the diffusion properties of highly flexible biopolymers such as IDPs. Likewise, recent empirical schemes that exploit the relationship between the small-angle x-ray scattering-informed conformational ensembles of IDPs and the respective experimental Dtr values also prove to be problematic. In this sense, the first-principle calculations of Dtr from the MD simulations, such as demonstrated in this work, should provide a useful benchmark for future efforts in this area.

X-ray Crystallography Module in MD Simulation Program Amber 2023. Refining the Models of Protein Crystals.

The MD simulation package Amber offers an attractive platform to refine crystallographic structures of proteins: (i) state-of-the-art force fields help to regularize protein coordinates and reconstruct the poorly diffracting elements of the structure, such as flexible loops; (ii) MD simulations restrained by the experimental diffraction data provide an effective strategy to optimize structural models of protein crystals, including explicitly modeled interstitial solvent as well as crystal contacts. Here, we present the new crystallography module xray, released as a part of the Amber 2023 package. This module contains functions to calculate and scale structure factors (including the contributions from bulk solvent), evaluate the maximum-likelihood-type crystallographic potential, and compute its derivative forces. The X-ray functionality of Amber no longer relies on external dependencies so that the full advantage of GPU acceleration can be taken. This makes it possible to refine in a short time hundreds of crystal models, including supercell models comprised of multiple unit cells. The new automated Amber-based refinement procedure leads to an appreciable improvement in Rfree (in some cases, by as much as 0.067) as well as MolProbity scores.

Conformational and Interaction Landscape of Histone H4 Tails in Nucleosomes Probed by Paramagnetic NMR Spectroscopy.

The fundamental repeat unit of chromatin, the nucleosome, consists of approximately 147 base pairs of double-stranded DNA and a histone protein octamer containing two copies each of histones H2A, H2B, H3, and H4. Each histone possesses a dynamically disordered N-terminal tail domain, and it is well-established that the tails of histones H3 and H4 play key roles in chromatin compaction and regulation. Here we investigate the conformational ensemble and interactions of the H4 tail in nucleosomes by means of solution NMR measurements of paramagnetic relaxation enhancements (PREs) in recombinant samples reconstituted with 15N-enriched H4 and nitroxide spin-label tagged H3. The experimental PREs, which report on the proximities of individual H4 tail residues to the different H3 spin-label sites, are interpreted by using microsecond time-scale molecular dynamics simulations of the nucleosome core particle. Collectively, these data enable improved localization of histone H4 tails in nucleosomes and support the notion that H4 tails engage in a fuzzy complex interaction with nucleosomal DNA.

Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.

The Role of Rotational Motion in Diffusion NMR Experiments on Supramolecular Assemblies: Application to Sup35NM Fibrils.

Pulsed-field gradient (PFG) NMR is an important tool for characterization of biomolecules and supramolecular assemblies. However, for micrometer-sized objects, such as amyloid fibrils, these experiments become difficult to interpret because in addition to translational diffusion they are also sensitive to rotational diffusion. We have constructed a mathematical theory describing the outcome of PFG NMR experiments on rod-like fibrils. To test its validity, we have studied the fibrils formed by Sup35NM segment of the prion protein Sup35. The interpretation of the PFG NMR data in this system is fully consistent with the evidence from electron microscopy. Contrary to some previously expressed views, the signals originating from disordered regions in the fibrils can be readily differentiated from the similar signals representing small soluble species (e.g. proteolytic fragments). This paves the way for diffusion-sorted NMR experiments on complex amyloidogenic samples.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-µs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

What Drives 15N Spin Relaxation in Disordered Proteins? Combined NMR/MD Study of the H4 Histone Tail.

Backbone (15N) NMR relaxation is one of the main sources of information on dynamics of disordered proteins. Yet, we do not know very well what drives 15N relaxation in such systems, i.e., how different forms of motion contribute to the measurable relaxation rates. To address this problem, we have investigated, both experimentally and via molecular dynamics simulations, the dynamics of a 26-residue peptide imitating the N-terminal portion of the histone protein H4. One part of the peptide was found to be fully flexible, whereas the other part features some transient structure (a hairpin stabilized by hydrogen bonds). The following motional modes proved relevant for 15N relaxation. 1) Sub-picosecond librations attenuate relaxation rates according to S2 ∼0.85-0.90. 2) Axial peptide-plane fluctuations along a stretch of the peptide chain contribute to relaxation-active dynamics on a fast timescale (from tens to hundreds of picoseconds). 3) φ/ψ backbone jumps contribute to relaxation-active dynamics on both fast (from tens to hundreds of picoseconds) and slow (from hundreds of picoseconds to a nanosecond) timescales. The major contribution is from polyproline II (PPII) ↔ ß transitions in the Ramachandran space; in the case of glycine residues, the major contribution is from PPII ↔ (ß) ↔ rPPII transitions, in which rPPII is the mirror-image (right-handed) version of the PPII geometry, whereas ß geometry plays the role of an intermediate state. 4) Reorientational motion of certain (sufficiently long-lived) elements of transient structure, i.e., rotational tumbling, contributes to slow relaxation-active dynamics on ∼1-ns timescale (however, it is difficult to isolate this contribution). In conclusion, recent advances in the area of force-field development have made it possible to obtain viable Molecular Dynamics models of protein disorder. After careful validation against the experimental relaxation data, these models can provide a valuable insight into mechanistic origins of spin relaxation in disordered peptides and proteins.

Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c-Crk N-SH3 Domain and Peptide Sos.

In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014 , 53 , 6473 ), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide-protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain (15)N relaxation rates, as well as binding shifts and (1)H(N) temperature coefficients. In addition, it was also modeled via a series of ∼5 µs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide "dances" over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due to its (loose) association with the protein. Note that spin relaxation data are indispensable in determining the dynamic status of the peptide. Such data can be properly modeled only on a basis of bona fide MD simulations, as shown in our study. We anticipate that in future the field will move away from the ensemble view of protein disorder and toward more sophisticated MD models. This will require further optimization of force fields, aimed specifically at disordered systems. Efforts in this direction have been recently initiated by several research groups; the empirical salt-bridge correction proposed in our work falls in the same category. MD models obtained with the help of suitably refined force fields and guided by experimental NMR data will provide a powerful insight into an intricate world of disordered biomolecules.

Role of electrostatic interactions in binding of peptides and intrinsically disordered proteins to their folded targets. 1. NMR and MD characterization of the complex between the c-Crk N-SH3 domain and the peptide Sos.

Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured ¹5N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 µs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions.

(1)H-(15)N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2NxHz or 2NxHx, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on Nx coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, Nx mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization (1)H-to-(15)N transfer. If CP element is designed such as to lock both (1)H(N) and water magnetization, the following transfer is effected: [Formula: see text] Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, kex ~ 100 s(-1), CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain H(ε)N(ε) groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using (13)C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as kex = 620 s(-1). In contrast, INEPT transfer completely fails in this regime.

Simulating diffraction photographs based on molecular dynamics trajectories of a protein crystal: a new option to examine structure-solving strategies in protein crystallography.

A molecular dynamics (MD)-based pipeline has been designed and implemented to emulate the entire process of collecting diffraction photographs and calculating crystallographic structures of proteins from them. Using a structure of lysozyme solved in-house, a supercell comprising 125 (5 × 5 × 5) crystal unit cells containing a total of 1000 protein molecules and explicit interstitial solvent was constructed. For this system, two 300 ns MD trajectories at 298 and 250 K were recorded. A series of snapshots from these trajectories were then used to simulate a fully realistic set of diffraction photographs, which were further fed into the standard pipeline for structure determination. The resulting structures show very good agreement with the underlying MD model not only in terms of coordinates but also in terms of B factors; they are also consistent with the original experimental structure. The developed methodology should find a range of applications, such as optimizing refinement protocols to solve crystal structures and extracting dynamics information from diffraction data or diffuse scattering.

Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD.

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.

Microsecond time-scale conformational exchange in proteins: using long molecular dynamics trajectory to simulate NMR relaxation dispersion data.

With the advent of ultra-long MD simulations it becomes possible to model microsecond time-scale protein dynamics and, in particular, the exchange broadening effects (R(ex)) as probed by NMR relaxation dispersion measurements. This new approach allows one to identify the exchanging species, including the elusive "excited states". It further helps to map out the exchange network, which is potentially far more complex than the commonly assumed 2- or 3-site schemes. Under fast exchange conditions, this method can be useful for separating the populations of exchanging species from their respective chemical shift differences, thus paving the way for structural analyses. In this study, recent millisecond-long MD trajectory of protein BPTI (Shaw et al. Science 2010, 330, 341) is employed to simulate the time variation of amide (15)N chemical shifts. The results are used to predict the exchange broadening of (15)N lines and, more generally, the outcome of the relaxation dispersion measurements using Carr-Purcell-Meiboom-Gill sequence. The simulated R(ex) effect stems from the fast (~10-100 µs) isomerization of the C14-C38 disulfide bond, in agreement with the prior experimental findings (Grey et al. J. Am. Chem. Soc. 2003, 125, 14324).

Very large residual dipolar couplings from deuterated ubiquitin.

Main-chain (1)H(N)-(15)N residual dipolar couplings (RDCs) ranging from approximately -200 to 200 Hz have been measured for ubiquitin under strong alignment conditions in Pf1 phage. This represents a ten-fold increase in the degree of alignment over the typical weakly aligned samples. The measurements are made possible by extensive proton-dilution of the sample, achieved by deuteration of the protein with partial back-substitution of labile protons from 25 % H(2)O / 75 % D(2)O buffer. The spectral quality is further improved by application of deuterium decoupling. Since standard experiments using fixed-delay INEPT elements cannot accommodate a broad range of couplings, the measurements were conducted using J-resolved and J-modulated versions of the HSQC and TROSY sequences. Due to unusually large variations in dipolar couplings, the trosy (sharp) and anti-trosy (broad) signals are often found to be interchanged in the TROSY spectra. To distinguish between the two, we have relied on their respective (15)N linewidths. This strategy ultimately allowed us to determine the signs of RDCs. The fitting of the measured RDC values to the crystallographic coordinates of ubiquitin yields the quality factor Q = 0.16, which confirms the perturbation-free character of the Pf1 alignment. Our results demonstrate that RDC data can be successfully acquired not only in dilute liquid crystals, but also in more concentrated ones. As a general rule, the increase in liquid crystal concentration improves the stability of alignment media and makes them more tolerant to variations in sample conditions. The technical ability to measure RDCs under moderately strong alignment conditions may open the door for development of alternative alignment media, including new types of media that mimic biologically relevant systems.

Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber.

A procedure has been developed for the refinement of crystallographic protein structures based on the biomolecular simulation program Amber. The procedure constructs a model representing a crystal unit cell, which generally contains multiple protein molecules and is fully hydrated with TIP3P water. Periodic boundary conditions are applied to the cell in order to emulate the crystal lattice. The refinement is conducted in the form of a specially designed short molecular-dynamics run controlled by the Amber ff14SB force field and the maximum-likelihood potential that encodes the structure-factor-based restraints. The new Amber-based refinement procedure has been tested on a set of 84 protein structures. In most cases, the new procedure led to appreciably lower R free values compared with those reported in the original PDB depositions or obtained by means of the industry-standard phenix.refine program. In particular, the new method has the edge in refining low-accuracy scrambled models. It has also been successful in refining a number of molecular-replacement models, including one with an r.m.s.d. of 2.15 Å. In addition, Amber-refined structures consistently show superior MolProbity scores. The new approach offers a highly realistic representation of protein-protein interactions in the crystal, as well as of protein-water interactions. It also offers a realistic representation of protein crystal dynamics (akin to ensemble-refinement schemes). Importantly, the method fully utilizes the information from the available diffraction data, while relying on state-of-the-art molecular-dynamics modeling to assist with those elements of the structure that do not diffract well (for example mobile loops or side chains). Finally, it should be noted that the protocol employs no tunable parameters, and the calculations can be conducted in a matter of several hours on desktop computers equipped with graphical processing units or using a designated web service.

Effect of rotation in NMR diffusion experiments on micron-sized particles: A generalized theoretical treatment.

We have recently developed an analytical framework to interpret the results of pulsed field gradient (PFG) NMR experiments on solution samples of micron-sized amyloid fibrils [Angew. Chem. Int. Ed. 60 (2021) 15445-15451. https://doi.org/10.1002/anie.202102408]. Here we generalize this result by reporting a rigorous theoretical model of such experiments, implemented in a form of efficient computational scheme. In particular, the new treatment fully accounts for the anisotropy of fibrils' translational diffusion and takes into consideration the finite length of the gradient pulses. The results hold not only for the historic spin-echo sequence, but also for the widely used stimulated echo experiment. We have found that fibrils' rotation can attenuate the echo by a factor comparable with that of translation. However, contrary to some recent claims, the rotational mechanism cannot lead to an apparent fast-diffusion situation.

Motion of a disordered polypeptide chain as studied by paramagnetic relaxation enhancements, 15N relaxation, and molecular dynamics simulations: how fast is segmental diffusion in denatured ubiquitin?

Molecular dynamics (MD) simulations have been widely used to analyze dynamic conformational equilibria of folded proteins, especially in relation to NMR observables. However, this approach found little use in the studies of disordered proteins, where the sampling of vast conformational space presents a serious problem. In this paper, we demonstrate that the latest advances in computation technology make it possible to overcome this limitation. The experimentally validated (calibrated) MD models allow for new insights into structure/dynamics of disordered proteins. As a test system, we have chosen denatured ubiquitin in solution with 8 M urea at pH 2. High-temperature MD simulations in implicit solvent have been carried out for the wild-type ubiquitin as well as MTSL-tagged Q2C, D32C, and R74C mutants. To recalibrate the MD data (500 K) in relation to the experimental conditions (278 K, 8 M urea), the time axes of the MD trajectories were rescaled. The scaling factor was adjusted such as to maximize the agreement between the simulated and experimental (15)N relaxation rates. The resulting effective length of the trajectories, 311 µs, ensures good convergence properties of the MD model. The constructed MD model was validated against the array of experimental data, including additional (15)N relaxation parameters, multiple sets of paramagnetic relaxation enhancements (PREs), and the radius of gyration. In each case, a near-quantitative agreement has been obtained, suggesting that the model is successful. Of note, the MD-based approach rigorously predicts the quantities that are inherently dynamic, i.e., dependent on the motional correlation times. This cannot be accomplished, other than in empirical fashion, on the basis of static structural models (conformational ensembles). The MD model was further used to investigate the relative translational motion of the MTSL label and the individual H(N) atoms. The derived segmental diffusion coefficients proved to be nearly uniform along the peptide chain, averaging to D = 0.49-0.55 × 10(-6) cm(2)/s. This result was verified by direct analysis of the experimental PRE data using the recently proposed Ullman-Podkorytov model. In this model, MTSL and H(N) moieties are treated as two tethered spheres undergoing mutual diffusion in a harmonic potential. The fitting of the experimental data involving D as a single adjustable parameter leads to D = 0.45 × 10(-6) cm(2)/s, in good agreement with the MD-based analyses. This result can be compared with the range of estimates obtained from the resonance energy transfer experiments, D = 0.2-6.0 × 10(-6) cm(2)/s.

Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a measure of domain-domain cooperativity, thus identifying the situations where two domains transiently interact with each other or become coupled through a partially structured linker.

Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics.

Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.