T.A. Holak

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing.

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.

Improved strategies for the determination of protein structures from NMR data: The solution structure of acyl carrier protein

The hybrid method that combines the early stages of a distance geometry program with simulated annealing in the presence of NMR constraints was optimized to obtain structures fully consistent with the observed NMR data. This was achieved by using more restrictive bounds of the NOE constraints than those usually used in the literature and by grouping the NOEs into classes dependent on the quality of the experimental NOE data. The ‘floating’ stereospecific assignment introduced at the simulated annealing stage of the calculations further improved the definition of the local conformation. An improved sampling and convergence property of the hybrid method was obtained by means of fitting the substructure obtained from the distance geometry program to different conformations. Compared to the standard hybrid methods, this procedure gave superior structures for a 77 amino acid protein, acyl carrier protein from Escherichia coli.

Nuclear magnetic resonance solution and X-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.

A comparison of the solution nuclear magnetic resonance (n.m.r.) structures of squash trypsin inhibitor from seeds of the squash Cucurbita maxima with the X-ray structure of a trypsin complex of the inhibitor shows that the n.m.r. and X-ray structures are similar in terms of the global folding and secondary structure. The average atomic root-mean-square difference between the 36 n.m.r. structures on the one hand and the X-ray structure is 0.96 A for the backbone atoms and 1.95 A for all heavy atoms. The n.m.r. and X-ray structures exhibit extremely similar conformations of the primary proteinase binding loop. Despite the overall similarity, there are small differences between the mean computed structure and the X-ray structure. The n.m.r. structures have slightly different positions of the segments from residues 16 to 18, and 24 and 25. The n.m.r. results show that the inclusion of stereospecific assignments and precise distance constraints results in a significant improvement in the definition of the n.m.r. structure, making possible a detailed analysis of the local conformations in the protein.

CAP2, cyclase-associated protein 2, is a dual compartment protein.

Abstract.Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

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.

Homonuclear three-dimensional NOE-NOE nuclear magnetic resonance/spectra for structure determination of proteins in solution☆

The solution structures of two proteins (CMTI-I, a trypsin inhibitor from Cucurbita maxima, and hisactophilin, an actin binding protein of 118 amino acids) have been determined based on the NOE data derived solely from the homonuclear 3D NOE-NOE magnetic resonance spectroscopy. Two different approaches for extraction of the structural information from the 3D NOE-NOE experiment were tested. One approach was based on the transformation of the 3D intensities into distance constraints. In the second, and more robust approach, the 3D NOE intensities were used directly in structure calculations, without the need to transform them into distance constraints. A new 2D potential function representing the 3D NOE-NOE intensity was developed and used in the simulated annealing protocol. For CMTI-I, a comparison between structures determined with the 3D NOE-NOE method and various 2D NOE approaches was carried out. The 3D data set allowed better definition of the structures than was previously possible with the 2D NOE procedures that used the isolated two-spin approximation to derive distance information.

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.

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.