A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance.

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on (15)N, natural abundance (13)C samples. The experiment exploits the fact that transfers of magnetization from (15)N to (13)CO and from (15)N to (13)C(alpha) (and back) proceed independently for samples that are not enriched in (13)C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM (15)N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.

Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in (13)C-labeled and fractionally (2)H-enriched proteins in solution.

New pulse sequences are presented for the measurement of the relaxation of deuterium double quantum, quadrupolar order, and transverse antiphase magnetization in (13)CH(2)D methyl groups of (15)N-, (13)C-labeled, fractionally deuterated proteins. Together with previously developed experiments for measuring deuterium longitudinal and transverse decay rates [Muhandiram, D. R.; Yamazaki, T.; Sykes, B. D.; Kay, L. E. J. Am. Chem. Soc. 1995, 117, 11536], these schemes allow measurement of the five unique decay constants of a single deuteron, providing an unprecedented opportunity to investigate side-chain dynamics in proteins. All five deuterium relaxation rates have been measured for deuterons in the methyl groups of the B1 immunoglobulin binding domain of peptostreptococcal protein L and the N-terminal SH3 domain from the protein drk. Since values of the spectral density function at only three different frequencies contribute to the five relaxation rates, the self-consistency of the relaxation data is readily established. Very good agreement is obtained between calculated parameters describing the amplitudes and time scales of motion when different subsets of the relaxation data are employed.

Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques.

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride.

Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy.

The use of a short, three-residue Cu(2+)-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu(2+) can be converted into distance restraints and used for structure refinement by using a new routine, PMAG, that has been written for the structure calculation program CNS. The utility of the approach is demonstrated with an application to ATCUN-tagged ubiquitin. Excellent agreement between measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.

Flexibility and ligand exchange in a buried cavity mutant of T4 lysozyme studied by multinuclear NMR.

The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the wild-type protein by measuring the NMR spin relaxation rates of backbone amide and side chain methyl nuclei. Many residues surrounding the cavity were found to be affected by a chemical exchange process with a rate of 1500 +/- 200 s(-1), which is quenched upon addition of saturating amounts of the ligand xenon. The relationship between the structure, dynamics, and energetics of the T4 lysozyme mutant is discussed.

Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes.

Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even polyproline stretches. The lack of amide protons in such regions complicates assignment, since 1HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the protein Crk with a region containing 9 prolines in a 14 amino acid sequence, as well as a WW domain that interacts with a proline-rich target. A modified version of the HACAN pulse scheme, originally described by Bax and co-workers [Wang et al. (1995) J. Biomol. NMR, 5, 376-382], and an experiment which correlates the intra-residue 1Halpha, 13Calpha/13Cbeta chemical shifts with the 15N shift of the subsequent residue are presented and applied to the two systems listed above, allowing sequential assignment of the molecules.

Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A 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.

Solution structure of a TBP-TAF(II)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP.

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three alpha helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: a role for N-terminal residues in tandem binding.

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H, 15N, 13C labelled intact and (des2-7) trp repressor (delta 2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound delta 2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.

Correlation between binding and dynamics at SH2 domain interfaces.

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 Cgamma1 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.

Pulse schemes for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings in 15N,13C uniformly labeled proteins.

Pulse sequences are presented for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings for all C gamma containing residues in 15N,13C uniformly labeled proteins. The methods described are based on quantitative J correlation spectroscopy pioneered by Bax and co-workers [Bax et. al. (1994) Methods Enzymol., 239, 79-105]. The combination of 3JC'C gamma and 3JNC gamma scalar coupling constants allows the assignment of discrete rotameric states about the chi 1 torsion angle in cases where such states exist or, alternatively, facilitates the establishment of noncanonical chi 1 conformations or the presence of rotameric averaging. The methods are applied to a 1.5 mM sample of staphylococcal nuclease.

Specific (15)N, NH correlations for residues in(15) N, (13)C and fractionally deuterated proteins that immediately follow methyl-containing amino acids.

A triple-resonance pulse scheme is described which records(15)N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a(15)N, (13)C and fractionally deuterated proteinsample and selects for CH(2)D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone (15)N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.

Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex.

The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.

Structure and dynamics of a CheY-binding domain of the chemotaxis kinase CheA determined by nuclear magnetic resonance spectroscopy.

The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals. Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which binds to the downstream targets of phosphorylation, the response regulators CheY and CheB. This protein fragment contains the CheY-binding domain flanked on each side by regions that correspond to domain linkers in the intact protein. The structure of the domain was determined from 1429 restraints derived from heteronuclear multidimensional NMR experiments. Hybrid distance geometry--dynamical simulated annealing methods were used to calculate a family of structures that satisfy the experimental distance restraints and torsion angle restraints. The root mean square deviation of the 69 ordered residues in the domain is 0.52 A for the backbone heavy atoms and 0.99 A for all heavy atoms. The residues that have been implicated as important for CheY binding form a face consisting of several partially buried hydrophobic residues, framed by charged residues. The dynamic properties of this protein fragment were measured and analyzed using both isotropic and anisotropic models of molecular motion. The linker regions are very flexible and disordered, as evidenced by the very dynamics properties as compared to the CheY-binding domain. The CheY-binding domain of CheA is structurally similar to the histidine-containing phosphocarrier, HPr, which is a protein involved in the phosphoenolpyruvate:sugar phosphotransferase (PTS) pathway. This structural similarity suggests a possible evolutionary relationship of the PTS and chemotaxis pathways.

Correlation between dynamics and high affinity binding in an SH2 domain interaction.

Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.

Solution structure of a cellulose-binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy.

Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.