Christian Cieslar

Practical and theoretical aspects of three-dimensional homonuclear Hartmann-Hahn-nuclear overhauser enhancement spectroscopy of proteins

The practical and theoretical aspects of three-dimensional homonuclear HartmannHahn-nuclear Overhauser enhancement spectroscopy (31) HOHAHA-NOESY) are presented and illustrated using the protein α1-purothionin as an example. A number of sequences are proposed with frequency selection either in the F1 dimension only or in both the F1 and the F2 dimensions, and their relative merits are discussed, particularly with respect to spectral resolution and measurement time. In addition, the nature of the various signals arising in 3D homonuclear spectroscopy, methods of evaluation of 3D HOHAHA-NOESY spectra, and the expected patterns of 3D cross peaks for different secondary structure elements in proteins are considered.

Three-dimensional homonuclear Hartmann-Hahn-nuclear Overhauser enhancement spectroscopy in H2O and its application to proteins

The potential of three-dimensional NMR spectroscopy to extend the methodology of protein structure determination by NMR has recently been demonstrated ( 2 ) . Peaks, generally referred to as relayed peaks in 2D NMR, are spread out into 3D frequency space, thereby reducing the extent of spectral overlap. Because the location of a 3D cross peak in 3D space is described by the chemical shifts of three different spins, i,j, k, the resolution of a 3D spectrum is superior to that of the corresponding 2D relayed spectrum. The key to protein structure determination by NMR lies in the identification of through-bond and through-space connectivities (2, 3). Particularly important are those involving the exchangeable NH protons. Hence it is necessary to work in H20. In our previous paper (I ) we presented a 3D nuclear Overhauser enhancement (NOESY)-homonuclear Hartmann-Hahn (HOHAHA) spectrum of the protein (Ye -purothionin in 90% H20. In this particular sequence, suppression of the water resonance other than by irradiation is technically very difficult, as well as prohibitive in measurement time due to the extensive phase cycling required (4). As a result, many correlations involving C”H protons that resonate in the vicinity of the water resonance are eliminated and signals of rapidly exchanging NH protons are abol ished by transfer of saturation. To overcome these problems, we have designed a novel 3D HOHAHA-NOESY pulse sequence in which the water resonance is suppressed using a jump-return read pulse (5) and frequency selection in the second dimension is achieved using a semiselective z filter (6). The superiority of this sequence over the previous one ( I ) is illustrated using (Y, -purothionin as an example. General schemes for 3D NMR spectroscopy consist of combining two 2D NMR techniques [e.g., COSY ( 7), NOESY (8), HOHAHA (9, IO)] leaving out the detection period of the first experiment and the preparation period of the second (I, II, 12). To reduce the size of the 3D data matrix and yet retain sufficiently well-resolved spectra, the two evolution times tl and t2 may be preceded by semiselective soft pulses to reduce the spectral widths in the first (Fl) and second (F2) dimensions (I Z-13).

Computer-assisted assignment of multidimensional NMR spectra of proteins: Application to 3D NOESY-HMQC and TOCSY-HMQC spectra

SummaryAn algorithm based on the technique of combinatorial minimization is used for the semi-automated assignment of multidimensional heteronuclear spectra. The program (ALFA) produces the best assignment compatible with the available input data. Even partially misleading or missing data do not seriously corrupt the final assignment. Ambiguous sequences of the possible assignment and all alternatives are indicated. The program can also use additional non-spectroscopic data to assist in the assignment procedure. For example, information from the X-ray structure of the protein and/or information about the secondary structure can be used. The assignment procedure was tested on spectra of mucous trypsin inhibitor, a protein of 107 residues.

Recognition of secondary-structure elements in 3D TOCSY-NOESY spectra of proteins. Interpretation of 3D cross-peak amplitudes

Abstract The amplitudes of selected 3D cross peaks in 3D TOCSY-NOESY spectra of proteins are shown to be characteristically different depending on the secondary structure. The sets of 3D cross peaks which make up the pattern for tight turns, β sheets, and α helices are given together with an estimate of the integral amplitude for each of the cross peaks obtained by a series development of the Hamiltonian of lowest order. A 3D TOCSY-NOESY spectrum of BPTI with a comparably short TOCSY mixing time was recorded and the volume integrals of the 3D cross peaks relevant for the recognition of the secondary-structure elements were compared to the amplitudes expected from a lowest order approximation.

Efficient methods for obtaining phase-sensitive gradient-enhanced HMQC spectra

SummaryThree improved versions of the gradient-enhanced HMQC experiment are presented which yield phase-sensitive spectra with increased sensitivity compared to the recently described field-gradient HMQC schemes. The first method uses a complex linear back-prediction in order to generate the FIDs at the t1=0. With this approach, refocusing pulses on the heteronucleus are not necessary. The sequence is especially useful for larger proteins with short relaxation times for the coherences that evolve during t1. In the other two methods lower and shorter gradient pulses or asymmetric gradients are used to optimize sensitivity.

3D TOCSY-TOCSY processing using linear prediction, as a potential technique for automated assignment

The assignment of the NMR spectra of proteins is the routine step in their structure determination (1, 2) and thus any approach to simplify or automate the evaluation of the spectra should be welcome. The first step in the assignment procedure is the proper recognition of the spin systems. This is commonly performed by 2D spectroscopy such as COSY, TOCSY, or relayed COSY (3-7) experiments and leads to a list of chemical shifts attributed to the protons involved in the coupling networks of the individual amino acids. There are already some approaches to automate this process (8-23), of which those based on “pattern recognition” in E.COSY (24) or z.COSY (25) spectra are the most prominent ones. The advantage of the latter procedure is the generality of the approach; i.e., any spin system may be recognized and the coupling network may be fully described. For proteins, this involves the recognition of the type of amino acid and the assignment of the resonances as NH, C,H, and CBH. Alternative approaches to the assignment of the spin systems are based on TOCSY and NOESY spectra which allow the collection of all chemical shifts of one residue, but may not always allow the correct assignment of the collected chemical shifts to the actual protons of the residue. Any approach based on 2D spectroscopy alone may suffer from the severe overlap of cross peaks usually observed in the aliphatic region of the spectra and may thus prevent the analysis of complicated coupling networks. Although the information contained in the relayed peaks of TOCSY or relayed spectra may help in overcoming ambiguities in some cases, the principal problem persists, especially as the peak-picking routines may no longer work due to overlap. For this reason, and because it is important to know the frequencies of the intermediate spins for the automated assignment, the consequent use of techniques yielding relayed peaks involves a three-dimensional Fourier transformation to resolve the overlap and to reveal the frequency of the intermediate spin (26-30). For the TOCSY technique, this can be achieved in a trivial manner by inserting an additional evolution time in the middle of the spin-lock period. Apart from the reduced amount of reasoning necessary by the computer there are a number of advantages which make the use of 3D TOCSY-TOCSY worthwhile: (i) peak-picking routines for 3D spectroscopy work more reliably, because lineshapes in three dimensions can be used as a criterion to recognize a peak; (ii) virtually no phase cycling is necessary and a three-dimensional spectrum with sufficient resolution can be recorded in a comparably short time; (iii)

A program for the evaluation of 3D spectra applied to the sequential assignment of BPTI utilizing 3D TOCSY-NOESY

In this Communication, we want to show that the use of homonuclear 3D spectroscopy is of advantage for the automatic assignment. The advantage of using cross peaks of 3D TOC-SY-NOESY spectra lies in the reduction of the number of possible nodes due to the better characterization of the connectivities

1H NMR assignments of sidechain conformations in proteins using a high-dimensional potential in the simulated annealing calculations

A high‐dimensional potential representing distance constraints for stereospecifically assignable diastereotopic proton or methyl pairs was incorporated into the dynamical simulated annealing protocol to calculate structures with stereospecifically determined sidechain conformations. The protocol is tested on nuclear magnetic resonance cross‐relaxation data of a trypsin inhibitor from squash seeds, CMTI‐I, and compared with two other methods of stereospecific assignment, the floating chirality and coupling constant methods. There is good agreement between the three methods in predicting the same stereospecific assignments. Because the high‐dimensional potential uses more relaxed absolute distance constraints and also takes into account the relative distance constraint patterns, it avoids possible overinterpretation of the NOE data.

Automatic phasing of pure phase absorption two-dimensional NMR spectra

A number of procedures have been reported for the automatic phasing of onedimensional NMR spectra (1-8) but none for two-dimensional spectra. Unlike the phasing of 1 D spectra, optimal manual phasing of pure-phase absorption 2D spectra (9) can be a time consuming procedure. Typically, this is carried out by Fourier transforming the 2D spectrum with phases acquired from inspection of the 1 D transforms of the free induction decays of the first few increments, followed by further corrections in both the w1 and the w2 dimensions. The phase corrections for the latter are usually determined by inspection of a few selected rows and columns. It is therefore clearly desirable to develop a method for the fully automated phase correction of twodimensional NMR spectra. In this paper we present a simple and efficient solution to this problem. The spectrum to be phased is a 2D hypercomplex spectrum in which the diagonal of the spectrum lies exactly on the diagonal of the point matrix representing the spectrum. Every point of the hypercomplex matrix consists of four values, RR, RI, IR, and II, which are the real and imaginary parts of the wI and o2 dimensions, of which only RR is displayed. Phase correction is carried out by multiplying the original values(RR,..., II) by the matrices

Some practical aspects of B0 gradient pulses

SummaryPulsed field gradients used together with trim pulses may cause artifacts in NMR spectra that originate from partial refocusing of dephased magnetization. These effects can reduce the efficiency of solvent suppression. The duration of the trim and PFG pulses should be in the range in which refocusing is negligible. Background gradients due to bad shimming also interfere with the B0 field gradient pulses, producing gradient-recalled echoes that reduce the receiver gain for NMR experiments. The shim settings can be optimized using simple experiments, as described in this paper. Eddy currents that cannot be completely compensated by adjustments of preemphasis induce phase shifts in NMR signals. The decay constants for a given spectrometer setup can easily be measured. If the experiment does not allow for proper compensation delays, the phase of the pulses must be adjusted to compensate for these phase shifts.