Solvent content of protein crystals.

Small Angle X-Ray Scattering

Publisher Summary This chapter reviews theoretical models that may be constructed and equations that may be derived from them to understand the process of protein denaturation. Given that the native state is stable under physiological conditions, the question arises whether the effects of environmental changes on the equilibrium between native and denatured states can be predicted, so as to account for the loss of stability of the native state and the appearance of different denatured states under specified conditions. This question involves not the absolute values for the free energies and other thermodynamic parameters for denaturation processes, but the changes in these parameters, along with changes in environmental variables. These changes can be predicted semiquantitatively. Furthermore, one can account both for the products formed under different conditions and for the character of the transitions from native to denatured state, at least for the simple proteins that have been studied in detail.

Protein denaturation. C. Theoretical models for the mechanism of denaturation.

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had <2 A RMSD error for the mainchain N, C(alpha), C, and O atoms; the average accuracies were 0.59 +/- 0.05, 1.16 +/- 0.10, and 2.61 +/- 0.16 A, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 A, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.

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Modeling of loops in protein structures.

Determination of the helix and beta form of proteins in aqueous solution by circular dichroism.

Structure of Hen Egg-White Lysozyme: A Three-dimensional Fourier Synthesis at 2 A Resolution

Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution.

Lysozyme was the second protein structure and the first enzyme structure to be solved by X-ray diffraction methods. The structure was published in 1965. Later work published in 1967 led to an explanation of the catalytic function of the enzyme and gave a vision of the explanatory power of structure for biological function. This chapter describes the crystals, the search for heavy-atom derivatives and the careful X-ray measurements made possible by the development of the linear diffractometer. By optimizing crystal mounting, by correcting for absorption effects and with considerable attention to radiation-damage effects implemented in the measurements and in the data-processing software, a complete data set to 2 A resolution was measured with remarkable precision. The precision of the measurements allowed anomalous-scattering effects to be introduced to help resolve the phase problem. The resulting electron-density map was immediately interpretable and the initial draft model building took about 1 month. The model was completed in time for it to form the centrepiece of Sir Lawrence Bragg's 75th birthday party at the Royal Institution on 31 March 1965. Many of the historical details of the data collection and structure determination are presented for the first time.

G. A. Mair

Papers

Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution.

How the structure of lysozyme was actually determined

Structural studies on the biological function of lysozyme