Ouwen Zhang

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

SummaryThe 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.

Spectral density function mapping using 15N relaxation data exclusively

SummaryA method is presented for the determination of values of the spectral density function, J(ω), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(ω) to be obtained at ω=0, ωN and 0.870ωH, where ωN and ωH are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H−15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(ω) can be mapped at ω=0, ωiN, ωjN, 0.870ωiH and 0.870ωjH, where ωiN, for example, is the 15N Larmor frequency for a spectrometer operating at i MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to trasverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N−NH bond vector are necessary, provided that dJ(ω)/dω is relatively constant between ω=ωH+ωN to ω=ωH−ωN. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium

SummaryA 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.

Triple-resonance NOESY-based experiments with improved spectral resolution: Applications to structural characterization of unfolded, partially folded and folded proteins

NMR-based structural studies of macromolecules focus to a large extent on the establishmentof interproton distances within the molecule based on the nuclear Overhauser effect (NOE).Despite the improvements in resolution resulting from multidimensional NMR experiments,the detailed characterization of disordered states of proteins or highly overlapped regions offolded molecules using current NMR methods remains challenging. A suite of triple-resonanceNOESY-type pulse schemes is presented which require uniform 15N and 13C labeling andmake use of the chemical shift dispersion of backbone 15N and 13C′ (carbonyl)resonances to increase the spectral resolution. In particular, for the case of partially folded andunfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C′resonances is comparable to that observed in folded states. Ambiguities that arise in theassignment of NOEs as a result of the severe chemical shift degeneracy in 1H and aliphatic13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C′either before or after the NOE mixing period. Applications of these methods to the study ofthe unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially foldedlarge fragment of staphylococcal nuclease (SNase), Δ131Δ, are presented. Inaddition, an application to folded SNase in complex with the ligands thymidine3′,5′-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignmentof NOEs between degenerate Hα protons or protons resonating close to water.

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 records15N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a15N, 13C and fractionally deuterated proteinsample and selects for CH2D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone 15N, 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.

Structural, Dynamic, and Folding Studies of SH2 and SH3 Domains

Recent advances in NMR methodology have made it a powerful approach for the study of biomolecular structure and dynamics (Bax and Grzesiek, 1993; Bax, 1994; Farrow et al., 1994a). In addition to NMR being a structural tool, as a solution spectroscopy it is exquisitely sensitive to dynamic processes - not only fast, low amplitude motions which can often be described by analysis of X-ray crystallographic B factors (Ringe and Petsko, 1985), but also slower, larger amplitude motions. These may include conformational exchange on millisecond time-scales or longer between states as dissimilar as folded and unfolded states of proteins and reflecting motions of tens of angstroms. We have exploited this distinguishing capability of NMR spectroscopy to describe the dynamic processes observed during structural studies of two isolated domains of signal transduction proteins, a Src Homology 2 (SH2) domain of phospholipase Cγ in complex with a phosphopeptide from the platelet-derived growth factor receptor (PDGFR) and an isolated Src Homology 3 (SH3) domain from the drosophila protein Drk1.