De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure.

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

Quantification of maximum-entropy spectrum reconstructions.

Maximum-entropy spectrum reconstruction derives much of its power from its nonlinearity. This nonlinearity causes difficulties in several contexts, however, including computation of multidimensional spectra and quantification of reconstructed spectra. We describe two methods for avoiding these difficulties: a "Constant-lambda" algorithm for performing row-wise reconstructions, which uses a fixed weighting of the entropy and the experimental constraint in the objective function, and in situ error analysis, for calibrating the nonlinearity. These methods are applied to data from quantitative J-correlation and relaxation experiments.

Distributed parallel processing for multidimensional maximum entropy reconstruction.

We have developed a two-dimensional maximum entropy spectrum reconstruction program designed to run in parallel on workstation clusters. Test reconstructions of planes extracted from a three-dimensional NMR data set indicate that the parallel speedup is nearly equal to the number of processors provided that the individual processors have comparable performance and that there are at least as many planes as processors. The program also works well in a typical laboratory setting consisting of heterogeneous workstations.

A structural study of the hydrophobic box region of lysozyme in solution using nuclear Overhauser effects.

Saturation of specific proton nuclear magnetic resonance (NMR) signals from residues in the hydrophobic box region of lysozyme (EC 3.2.1.17) has enabled negative nuclear Overhauser effects to be measured on the resonances of nearby protons. The assignments of resonances reported previously have been examined, and most have been confirmed. In conjunction with spin-decoupling methods, new assignments could be made so that assignments for some 70 resonances of 25 residues in lysozyme are now known. A high correlation was observed between the observed nuclear Overhauser effects and interproton distances calculated from cyrystallographic data. This indicates that the average structure of this region of lysozyme in the crystalline state is maintained in solution, that substantial populations of structures very different from this do not exist, and that the nuclear Overhauser technique can be applied in a straightforward manner to obtain structural data in solution at the 1-A level.

Vicinal coupling constants and protein dynamics.

The effects of motional averaging on the analysis of vicinal spin-spin coupling constants derived from proton NMR studies of proteins have been examined. Trajectories obtained from molecular dynamics simulations of bovine pancreatic trypsin inhibitor and of hen egg white lysozyme were used in conjunction with an expression for the dependence of the coupling constant on the intervening dihedral angle to calculate the time-dependent behavior of the coupling constants. Despite large fluctuations, the time-average values of the coupling constants are not far from those computed for the average structure in the cases where fluctuations occur about a single potential well. The calculated differences show a high correlation with the variation in the magnitude of the fluctuations of individual dihedral angles. For the cases where fluctuations involve multiple sites, large differences are found between the time-average values and the average structure values for the coupling constants. Comparison of the simulation results with the experimental trends suggests that side chains with more than one position are more common in proteins than is inferred from X-ray results. It is concluded that for the main chain, motional effects do not introduce significant errors where vicinal coupling constants are used in structure determinations; however, for side chains, the motional average can alter deductions about the structure. Accurately measured coupling constants are shown to provide information concerning the magnitude of dihedral angle fluctuations.

Solution structure of LSIII, a long neurotoxin from the venom of Laticauda semifasciata.

We report the sequence-specific proton assignments and solution structure of the long neurotoxin LSIII from the venom of Laticauda semifasciata determined by two- and three-dimensional 1H NMR. Input for structure calculations consisted of 497 NOE-derived distance restraints and 45 dihedral angle restraints obtained from J couplings. A two-particle-per-residue representation of protein structure was used to generate 200 initial structures which were then subjected to all-atom refinement by simulated annealing. Twenty-three final structures consistent with the experimental restraints were obtained; the average atomic RMS difference between the individual structures and the mean structure was 0.82 A for the backbone heavy atoms and 1.3 A for all heavy atoms (residues 1-26, 37-60). The main elements of regular secondary structure are a three-stranded antiparallel beta-sheet and three finger-like loops protruding from a globular core, consistent with previously reported structures of long neurotoxins. The end of the prominent loop II, which is involved in binding to acetylcholine receptor, is disordered relative to the rest of the molecule. A novel finding of this study is that the loop has a well defined local structure; this and other observations suggest this region moves as a rigid body. We propose that this motion is a heretofore unrecognized general feature of long neurotoxins, with specific consequences for binding to the acetylcholine receptor.

Fluctuations and averaging of proton chemical shifts in the bovine pancreatic trypsin inhibitor.

The effects of motional averaging on the aromatic ring-current contribution to the proton chemical shifts in proteins are examined. Atomic trajectories obtained from a 96-ps molecular dynamics simulation of the bovine pancreatic trypsin inhibitor are used in conjunction with the Johnson-Bovey model of ring-current shifts to calculate the time evolution of the proton chemical shifts. Although large high-frequency fluctuations are observed (often greater than +/- 1 ppm), the average shift values in most cases are close to those obtained from the average structure; for some protons, significant differences are found. The calculated trends are used to probe the relationship between the average structure, atomic motions, and observed values of the proton chemical shifts. It is concluded that chemical shift values are in general most sensitive to the average structure of the protein and, because of the averaging involved, cannot be used directly to probe the short time structural fluctuations.

Application of nonlinear sampling schemes to COSY-type spectra.

Nonlinear sampling along the t1 dimension is applied to COSY-type spectra. The sine dependence of the time domain signals for the cross peaks is matched by a nonlinear sampling scheme that samples most densely around the maximum of the sine function. Data are processed by maximum entropy reconstruction, using a modified implementation of the 'Cambridge' algorithm of Skilling and Bryan. The procedure is demonstrated for P.E. COSY spectra recorded on a cyclic hexapeptide and on a 126-residue domain of the protein villin. The number of t1 values in the nonlinearly sampled experiments was reduced by a factor of four compared to linear sampling. The sensitivity and resolution of the resulting spectra are comparable to those achieved by conventional methods. The method described can thus significantly reduce the measuring time for COSY-type spectra.

Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy.

The arrival of very high field magnets and cryogenic circuitries, and the development of relaxation-optimized pulse sequences have added powerful tools for increasing sensitivity and resolution in NMR studies of biomacromolecules. The potential of these advances is not fully realized in practice, however, since current experimental protocols do not permit sufficient data sampling for optimal resolution in the indirect dimensions. Here we analyze quantitatively how increasing resolution in indirect dimensions affects the S/N ratio and compare this with currently used sampling routines. Optimal resolution would require sampling up to approximately 3 R (2)(-1), and the S/N reaches a maximum at approximately 1.2 R (2)(-1). Currently used data acquisition protocols rarely sample beyond 0.4 R (2)(-1), and extending evolution times would result in prohibitively long experiments. We show that a general solution to this problem is to use non-uniform sampling, where only a small subset of data points in the indirect sampling space are measured, and possibly different numbers of transients are collected for different evolution times. Coupled with modern methods of spectrum analysis, this strategy delivers substantially improved resolution and/or reduced measuring times compared to uniform sampling, without compromising sensitivity. Higher resolution in the indirect dimensions will facilitate the use of automated assignment programs.

Does the maximum entropy method improve sensitivity?

Maximum entropy reconstruction has been used in several fields to produce visually striking reconstructions of positive objects (images, densities, spectra) from noisy, indirect measurements. In magnetic resonance spectroscopy, this technique is notable for its apparent noise suppression and its avoidance of the artifacts that affect discrete Fourier transform spectra of short (zero-extended) data records. In the general case where the length of the reconstructed spectrum exceeds that of the data record or where a convolution kernel is incorporated in the reconstruction, no known analytical solution to the reconstruction problem exists. Consequently, knowledge of the properties of maximum entropy reconstruction has been mainly anecdotal, based on a small selection of published reconstructions. However, in the limiting case where the lengths of the reconstructed spectrum and the data record are the same and a convolution kernel is not applied, the problem can be solved analytically. The solution has a simple structure that helps explain several commonly observed features of maximum entropy reconstructions--for example, the biases in the recovered intensities and the fact that noise near the baseline is more successfully suppressed than is noise superimposed on broad features in the spectrum. The solution also shows that the noise suppression offered by maximum entropy reconstruction could (in this special case) be equally well obtained by a "cosmetic" device: simply displaying the conventional Fourier transform reconstruction using a certain nonlinear plotting scale for the vertical (y) coordinate.

Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain.

Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Gal4. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.