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Dynamics are often critical for biomolecular function. Herein we explore the role of motion in driving the maturation process of pro-IL-18, a potent pro-inflammatory cytokine that is cleaved by caspases-1 and -4 to generate the mature form of the protein. An NMR dynamics study of pro-IL-18, probing time scales over 12 orders of magnitude and focusing on 1H, 13C, and 15N spin probes along the protein backbone and amino-acid side chains, reveals a plastic structure, with millisecond time scale dynamics occurring in a pair of β-strands, β1 and β*, that show large structural variations in a comparison of caspase-free and bound pro-IL-18 states. Fits of the relaxation data to a three-site model of exchange showed that the ground state secondary structure is maintained in the excited conformers, with the side chain of I48 that undergoes a buried-to-exposed conformational change in the caspase-free to -bound transition of pro-IL-18, sampling a more extensive range of torsion angles in one of the excited states characterized, suggesting partial unpacking in this region. Hydrogen exchange measurements establish the occurrence of an additional process, whereby strands β1 and β* locally unfold. Our data are consistent with a hierarchy of dynamic events that likely prime pro-IL-18 for facile caspase binding.

NMR spectroscopy is an important tool for the measurement of the electrostatic properties of biomolecules. To this point, paramagnetic relaxation enhancements (PREs) of 1H nuclei arising from nitroxide cosolutes in biomolecular solutions have been used to measure effective near-surface electrostatic potentials (ϕENS) of proteins and nucleic acids. Here, we present a gadolinium (Gd)-based NMR method, exploiting Gd chelates with different net charges, for measuring ϕENSvalues and demonstrate its utility through applications to a number of biomolecular systems. The use of Gd-based cosolutes offers several advantages over nitroxides for ϕENSmeasurements. First, unlike nitroxide compounds, Gd chelates enable electrostatic potential measurements on oxidation-sensitive proteins that require reducing agents. Second, the large electron spin quantum number of Gd (7/2) results in notably larger PREs for Gd chelates when used at the same concentrations as nitroxide radicals. Thus, it is possible to measure ϕENSvalues exclusively from + and – charged compounds even for highly charged biomolecules, avoiding the use of neutral cosolutes that, as we further establish here, limits the accuracy of the measured electrostatic potentials. In addition, the smaller concentrations of cosolutes required minimize potential binding to sites on macromolecules. Fourth, the closer proximity of the paramagnetic center and charged groups within Gd chelates, in comparison to the corresponding nitroxide compounds, enables more accurate predictions of ϕENSpotentials for cross-validation of the experimental results. The Gd-based method described here, thus, broadens the applicability of studies of biomolecular electrostatics using solution NMR spectroscopy.

Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42(Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.

Background Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. Methods 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ--proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. Results 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [[IC.sub.50]] of AMC release in intact cells ranged from 0.57 to 5.03 [micro]M), induced cell death in intact cells from leukemia and myeloma cell lines (mean [IC.sub.50] values for cell growth ranged from 0.94 to 3.85 [micro]M), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding [beta]5 proteasome subunit (mean [IC.sub.50] for cell death in the absence of bortezomib, wild-type THP1: 3.7 [micro]M, 95% confidence interval = 3.4 to 4.0 [micro]M; THP1/BTZ500:6.6 [micro]M, 95% confidence interval = 5.9 to 7.5 [micro]M). 5AHQ interacted with the [alpha] subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). Conclusions 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro. J Natl Cancer Inst 2010;102:1069-1082 DOI: 10.1093/jnci/djq198

ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure–function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen Neisseria meningitidis. Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.

Intrinsically disordered proteins (IDPs) or regions of intrinsic disorder in otherwise folded proteins (IDRs) play important roles in many different biological processes, including formation of biological condensates via liquid–liquid phase separation. NMR spectroscopy is a powerful tool for obtaining site-specific structural and dynamical information on IDPs/IDRs, and recent efforts have focused on the development of experiments for atomic-resolution studies of these molecules. These include triple-resonance experiments that are based on 13CO-direct detection of magnetization, exploiting increased sensitivity of cryogenically cooled probes. In order to evaluate the different classes of experiment for studies of IDRs or IDPs in both dilute and phase-separated environments, in particular at neutral and higher pHs where many of these proteins phase separate, we compared 13CO-detect versus 1Hα-detect experiments, showing that significant sensitivity gains are achieved via proton detection under the conditions of our experiments. A suite of 1Hα-detect experiments was subsequently developed for studies of IDPs/IDRs and applied to the dilute phase of a 103-residue disordered region of CAPRIN1 that phase separates at neutral pH. Residue-specific chemical shifts derived from our study enable the accurate prediction of the importance of the N-terminal Arg-containing region of this construct for promoting phase separation relative to other Arg-rich stretches of sequence, subsequently confirmed by mutagenesis. Our study emphasizes that the sequence positions of key residues can be a critical factor in controlling phase separation and highlights the unique role of NMR in establishing the relations between amino acid sequence and phase-separation propensity.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host–guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2′-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA–RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA–RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder–order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

Immunoglobulin light-chain amyloidosis is a protein aggregation disease that leads to proteinaceous deposits in a variety of organs in the body and, if untreated, ultimately results in death. The mechanisms by which light-chain aggregation occurs are not well understood. Here we have used solution NMR spectroscopy and biophysical studies to probe immunoglobulin variable domain λV6-57 VLaggregation, a process that appears to drive the degenerative phenotypes in amyloidosis patients. Our results establish that aggregation proceeds via the unfolded state. We identify, through NMR relaxation experiments recorded on the unfolded domain ensemble, a series of hotspots that could be involved in the initial phases of aggregate formation. Mutational analysis of these hotspots reveals that the region that includes K16-R24 is particularly aggregation prone. Notably, this region includes the site of the R24G substitution, a mutation that is found in variable domains of λ light-chain deposits in 25% of patients. The R24G λV6-57 VLdomain aggregates more rapidly than would be expected on the basis of thermodynamic stability alone, while substitutions in many of the aggregation-prone regions significantly slow down fibril formation.

Solvent exchange rates provide important information about the structural and dynamical properties of biomolecules. A large number of NMR experiments have been developed to measure such rates in proteins, the great majority of which quantify the buildup of signals from backbone amides after initial perturbation of water magnetization. Here we present a different approach that circumvents the main limitations that result from these classical hydrogen exchange NMR experiments. Building on recent developments that enable rapid recording of chemical exchange saturation transfer (CEST) pseudo-3D data sets, we describe a 15N-based CEST scheme for measurement of solvent exchange in proteins that exploits the one-bond 15N deuterium isotope shift. The utility of the approach is verified with an application to a 236 residue intrinsically disordered protein domain under conditions where it phase separates and a second application involving a mutated form of the domain that does not phase separate, establishing very similar hydrogen exchange rates for both samples. The methodology is well suited for studies of hydrogen exchange in any 15N-labeled biomolecule. A discussion of the merits of the CEST experiment in relation to the popular CLEANEX-PM scheme is presented.

Protein oligomerization in the cell has important implications for both health and disease, and an understanding of the mechanisms by which proteins can self-associate is, therefore, of critical interest. Initial stages of the oligomerization process can be hard to detect, as they often involve the formation of sparsely populated and transient states that are difficult to characterize by standard biophysical approaches. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we study the oligomerization of human profilin-1, a protein that regulates the polymerization of actin. We show that in solution and at millimolar concentrations profilin-1 is predominantly monomeric. However, fits of concentration-dependent relaxation data are consistent with the formation of a higher-order oligomer that is generated via a multistep process. Together with crystallographic data for profilin-2, a homologue of the protein studied here, our results suggest that profilin-1 forms a sparsely populated tetrameric conformer in solution.

The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of density maps and structural models that include those for novel particles of approximately 30 and 60 monomers. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.

•A pedagogical description of relaxation theory in the presence of a B1 field is presented.•Two different theoretical approaches found in the literature are considered.•The identity of the two approaches is shown.

Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a “high-resolution” picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 °C) ground state conformation and an excited state that is 2.0 kcal mol−1higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (ΔH = 7.1 kcal mol−1) and entropic (TΔS = 5.1 kcal mol−1, 25 °C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with β-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/β-cyclodextrin complex where the solution state is found to be ∼10° more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high-resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (∼17° closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (∼11°). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.

The global fold of maltose-binding protein in complex with the substrate β-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H^N-H^N, H^N-CH₃ and CH₃-CH₃ NOEs was recorded on ¹⁵N, ²H and ¹⁵N, ¹³C, ²H-labeled proteins with methyl protonation in Val, Leu, and Ile (C^δ1 only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 Å was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 Å, respectively. Direct refinement against one-bond ¹H^N-¹⁵N, ¹³C^α-¹³CO, ¹⁵N-¹³CO, two-bond ¹H^N-¹³CO and three-bond ¹H^N-¹³C^α dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 Å, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 Å. Copyright 2000 Academic Press

We have expanded our description of the energy landscape for folding of the SH3 domain of chicken α-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on 2H, 15N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native α-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.

The status of the poly(A) tail at the 3′-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3′-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with β-cyclodextrin has been determined on the basis of one-bond 15N-HN, 15N-13C′, 13Cα-13C′, two-bond 13C′-HN, and three-bond 13Cα-HN dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(±1)° domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5° uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins.

The phosphotyrosine-binding (PTB) domain of Numb, a protein involved in asymmetric cell division, has recently been shown to bind to the adapter protein Lnx through an LDNPAY sequence, to the Numb-associated kinase (Nak) through a sequence that does not contain an NPXY motif and to GP(p)Y-containing peptides obtained from library screening. We show here that these diverse peptide sequences bind with comparable affinities to the Numb PTB domain at a common binding site on the surface of the protein. The NMR structure of the Numb PTB domain in complex with a GPpY-containing peptide reveals a novel mechanism of binding with the peptide in a helical turn that does not hydrogen bond to the PTB domain β-sheet. These results suggest that PTB domains can potentially have multiple modes of peptide recognition and provide a structural basis from which the multiple functions of the Numb PTB domain during asymmetric cell division could arise.

A 4D TROSY-based triple resonance experiment, 4D-HNCOi-1CAi, is presented which correlates intra-residue 1HN, 15N, 13 Ca chemical shifts with the carbonyl (13C') shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (1HN,15N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCOi-1CAi often allows resolution of the ambiguity by linking the 13Ca and 13C' spins surrounding the (1HN,15N) pair. The experiment is demonstrated on a sample of 15N, 13C, 2 H labeled maltose binding protein in complex with ß-cyclodextrin that tumbles with a correlation time of 46 ns.

The N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (Fexch) and an unfolded state (Uexch) under non-denaturing buffer conditions. Using a 15N/2H-labeled sample, long range amide NOEs can be observed in the Uexchstate as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the Uexchand the guanidine denatured states. Calculations using the long range NOEs of the Uexchstate yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivoand whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five α-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 Å in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 Å for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

Protein recognition is a key determinant in regulating biological processes. Structures of complexes of interacting proteins provide significant insights into the mechanism of specific recognition. However, studies performed by modifying residues within a protein interface demonstrate that binding is not fully explained by these static pictures. Thus, structural data alone was not predictive of affinities in binding studies of phospholipase Cγ1 and Syp phosphatase SH2 domains with phosphopeptides. NMR relaxation experiments probing dynamics of methyl groups of these complexes indicate a correlation between binding energy and restriction of motion at the interfacial region responsible for specific binding.

A biosynthetic strategy has recently been developed for the production of 15N, 13C, 2H-labeled proteins using 1H3C-pyruvate as the sole carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (?2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a 15N, 13C, 2H, 1H3C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.

Essentially complete backbone and side-chain 1H, 15N and 13C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [15N]-and [15N, 13C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of 13C-chemical shift, amide hydrogen-bond exchange, and 15N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five a-helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.

A pulsed field gradient three-dimensional isotope-filtered 13C HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp-repressor, its natural abundance co-repressor, l-tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra- and intermolecular NOEs in the isotope-edited 3D 13C HMQC-NOESY spectrum of the complex.

Two new NMR experiments are presented for measuring side-chain dynamics in proteins. The first method, requiring 15N, 13C, ∼50% 2H-labeled protein, measures 2H T1and T1ρspin relaxation times at side-chain positions. A second experiment permits the straightforward measurement of 13C1H dipole-dipole cross-correlation relaxation rates at 13Cβpositions in 15N,13C-labeled molecules. An excellent correlation is observed between order parameters, describing the amplitude of motion at these sites, obtained on the basis of 2H relaxation and dipole-dipole cross-correlation relaxation rates. Together these experiments provide a powerful approach for selecting appropriate motional models. The methods are applied to study the side-chain motional properties of the N-terminal SH3 domain from the signaling protein drk.

The relation between order parameters derived from NMR spin relaxation experiments and the contribution to conformational entropy from ns-ps timescale bond vector dynamics is investigated by considering a number of simple models describing bond vector motion. In a few cases both classical and quantum mechanical derivations are included to establish the validity of obtaining order parameter-entropy relations using classical mechanics only. For these cases it is found that classical and quantum mechanical derivations give very similar results so long as the square of the order parameter of the bond vector is less than ~0.95. For a given change in order parameter, the change in conformational entropy is sensitive to the model employed, with the absolute value of the entropy change increasing with the number of degrees of freedom in the model. The entropy-order parameter profile calculated from a 1.12 ns molecular dynamics trajectory of fully hydratedEscherichia coliribonuclease HI is well fit using a simple expression based on a model assuming bond vector diffusion in a cone, suggesting that it may well be possible to extract meaningful entropy changes reflecting changes in ps-ns time scale motions from changes in NMR-derived order parameters. Contributions to the conformational entropy change associated with a folding-unfolding transition of an SH3 domain and calculated from changes in rapid N-H^Nbackbone dynamics are presented.

Comprehensive NOE results from detailed structural characterization of a 131 residue partially folded fragment of staphylococcal nuclease (Δ131Δ) made possible by NMR methods with improved resolution are presented. The resulting NOE patterns reflect sampling of both α and β regions of φ, ϕ conformational space, yet demonstrate significant preferences for both native-like and non-native-like turn and potentially helical conformations. Together with data from studies of the unfolded state of the drkN SH3 domain, NOE patterns observed for partially folded or unfolded proteins are summarized. It is surprising that few long-range NOEs were observed in Δ131Δ. The two longest-range NOEs are both native-like; one of these, an (i, i+5) NOE, provides evidence for a Schellman capping motif for helix termination. Many aliphatic-aliphatic and aliphatic-amide NOEs, which are not normally observed in folded proteins, were detected. We have ruled out significant contributions from spin-diffusion for a number of these NOEs and suggest that one source may be sampling of non-prolyl cispeptide bond configurations in the disordered state of Δ131Δ.

In the past several years a significant number of new multidimensional NMR methods have been developed to study molecular dynamics spanning a wide range of time scales. Applications involving a large number of biological systems have emerged and correlations with function established. Unique insights are obtained that are not available from structure alone, indicating the importance of dynamics studies for understanding function.

Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of¹H-¹H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpres sion of proteins in bacteria grown in²H₂O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C^αand >80% deuteration at C^βpositions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (γ2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.

An analytical description of the evolution of magnetization in InS spin systems (1 ≤n≤ 3) during the course of an adiabatic pulse applied on spin S is provided. Calculations show that multiple-quantum terms are created during the pulse and that the rate at which in-phase and antiphase I-spin magnetization components interchange during spin-echo-based pulse sequences is decreased relative to the case where a hard inversion pulse is substituted for the adiabatic pulse. This has important consequences for purging schemes making use of such frequency-swept pulses. Simulations demonstrate that the evolution of in-phase I magnetization is essentially independent ofn.

The T1-accordion COSY experiment has been applied to acyl carrier protein (ACP) to locate the divalent ion binding sites in the protein using the paramagnetic ion, Mn 2+, as a substitute for Ca 2+. Replacement with Mn 2+leads to an enhancement of proton spin-lattice ( T1) relaxation rates. These enhancements have a l/ r6, distance dependence that makes them extremely useful in structural analyses. Ion-proton distances ranging from 3.0 to 9.0 Å have been obtained from this experiment and subsequently used as constraints in the molecular mechanics module of AMBER to refine a protein structure.

Present analysis procedures for NMR structure determination of macromolecules presuppose fixed internuclear distances. Improvement of the precision of the requisite NOE information has stimulated the use of more quantitative distance constraints thus necessitating examination as to whether the assumption of a rigid model systematically biases the distance estimates. Analysis using the simple < r−6> dependence of NOE buildup rates seriously underestimates the correct distance for spatially proximal proton pairs having fluctuations comparable to those observed in X-ray temperature factor analysis. However, by calculating the proper generalized order parameter it is shown that for nuclei undergoing rapid isotropic uncorrelated fluctuations the effective distance is identical to the distance between the mean positions of the nuclei. Similar analysis of molecular dynamics simulation data from bovine pancreatic trypsin inhibitor indicates that the distance obtained from the generalized order parameter predicts the distance between the mean positions to within a few percent regardless of the degree of correlation of the pairwise motion for virtually all main chain and dynamically constrained side chain protons.

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6°C is 4.86 (±0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D∥/D⊥, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample.Based on the dynamic characteristics of TnC, two different levels of “fine tuning” of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (ΔΔSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 μM and 1.7 μM, respectively.In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Summary The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.

A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, and approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

An enhanced-sensitivity gradient 4D 15N, 13C-edited NOESY experiment is presented. Gradients are employed to suppress artifacts, eliminate the intense H2O signal and select for the coherence transfer pathway involving 15N magnetization. The latter use of the gradients results in a decrease in the number of phase cycle steps by a factor of two relative to the previously published 4D sequence (Kay et al., 1990) allowing for the recording of spectra with increased resolution per unit measuring time. Theoretical sensitivity enhancements of as much as a factor of $$\sqrt 2 $$ can be expected over the previously published sequence, neglecting the effects of relaxation and pulse imperfections.

Summary An improved version of the three-dimensional HCCH-COSY NMR experiment is described that correlates the resonances of geminal and vicinal proton pairs with the chemical shift of the13C nucleus attached to one of the protons. The experiment uses constant-time evolution of transverse13C magnetization which optimizes transfer of magnetization and thus improves the sensitivity of the experiment over the original scheme. The experiment is demonstrated for calmodulin complexed with a 26-residue peptide comprising the binding site of skeletal muscle myosin light chain kinase.

The backbone dynamics of both folded and unfolded states of staphylococcal nuclease (SNase) and the N-terminal SH3 domain from drk (drkN SH3) are studied at two different temperatures. A simple method for obtaining order parameters, describing the amplitudes of motion of bond vectors, from NMR relaxation measurements of both folded and unfolded proteins is presented and the data obtained for 15N-NH bond vectors in both the SNase and drkN SH3 systems analyzed with this approach. Using a recently developed theory relating the amplitude of bond vector motions to conformational entropy, the entropy change between the folded and unfolded forms of SNase is calculated on a per residue basis. It is noteworthy that the region of the molecule with the smallest entropy change includes those residues showing native-like structure in the unfolded form of the molecule, as established by NOE-based experiments. Order parameters of backbone 15N-NH bond vectors show significantly larger changes with temperature in the unfolded states of both proteins relative to the corresponding folded forms. The differential temperature dependence is interpreted in terms of differences in the heat capacities of folded and unfolded polypeptide chains. The contribution to the heat capacity of the unfolded chain from rapid 15N-NH bond vector motions is calculated and compared with estimates of the heat capacity of the backbone unit, -CHCONH-, obtained from calorimetric data. Methyl dynamics measured at 14 and 30°C establish that the amplitudes of side-chain motions in the folded SH3 domain are more sensitive to changes in temperature than the backbone dynamics, suggesting that over this temperature range side-chain ps to ns time-scale motions contribute more to the heat capacity than backbone motions for this protein.