Eaton E. Lattman

High Apparent Dielectric Constants in the Interior of a Protein Reflect Water Penetration

A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.

Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution

Crystals of sickle-cell deoxyhemoglobin were grown from solutions containing polyethylene glycol and citrate-phosphate buffer at a pH between 5 and 6. The crystals have the symmetry of the monoclinic space group P21, with a=63·28 A, b=184·19 A, c=52·84 A, and β=92·67°. The structure was determined by rotational and translational search procedures. Structure amplitudes and phases were calculated from the atomic co-ordinates of deoxy Hb§ A molecules appropriately positioned in the unit cell of the deoxy Hb S crystal. An Fo−Fo difference Fourier for the Hb S cristal was compouted at 5 A resolution. Portions of the Hb S molecules near the Val6β residues do not appear to be significantly different from the same portions of deoxy Hb A molecules crystallized in polyethylene glycol solutions at pH 7. In the Hb S crystal the molecular x axes enclose angle of less than 10° with the crystallographic a axis. The molecules are arranged in pairs of interlocking strands aligned with the a axis. The two strands in each pair are related approximately a 2-fold screw axis running between them longitudinally. Intermolecular contacts within each pair of strands involve Val6β and other residues that are believed to affect sickling interactions. Double strands, similar to those found in the Hb S crystal, can be incorporated into a fiber model that is consistent with available information on the structure of deoxy Hb S fibers in vivo.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.

Representation of phase probability distributions for simplified combination of independent phase information

ANDRES, K. (1968). Phys. Rev. 170, 614. BARRETT, C. S., MUELLER, M. H. & HITTERMAN, R. L. (1963). Phys. Rev. 129, 625. COPPENS, P. & HAMILTON, W. C. (1968). American Crystallographic Association Meeting, Buffalo, New York. Abstract G2. FISHER, E. S. & DEVER, D. (1968). Phys. Rev. 170, 607. HEATON, L., MUELLER, M. H. & HITTERMAN, R. L. (1968). American Crystallographic Association Meeting, Buffalo, New York. Abstract H 3. HOUGH, A., MARPLES, J. A. C., MORTIMER, M. J. & LEE, J. A. (1968). Phys. Letters, 27A, 222. Ross, J. W. & LAM, D. J. (1968). Phys. Rev. 165, 617. WILLIS, B. T. M. (1962). Pile Neutron Research in Physics, p. 455. Vienna: I.A.E.A. ZACHARIASEN, W. H. 1.1967). Acta Cryst. 23, 558. ZACHARIASEN, W. H. (1968a). Acta Cryst. A24, 212. ZACHARIASEN, W. H. (1968b). Acta Cryst. A24, 324.

Experimental pKa Values of Buried Residues: Analysis with Continuum Methods and Role of Water Penetration

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amines relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.

One-step evolution of a dimer from a monomeric protein

Deletion of six amino acids in a surface loop transforms staphylococcal nuclease from a monomeric protein into a very stable dimer (Kd<1×10−8M). A 2 Å X-ray crystal structure of the dimer (R=0.176) shows that the carboxy-terminal α-helix has been stripped from its normal position in one monomer and is now incorporated into the equivalent position on the adjoining monomer. This swapping creates an association interface of 2900 Å2. A second, smaller interface of 460 Å2 is also formed. The spontaneous exchange or swapping of secondary structural elements provides a simple pathway for the formation of large, stable protein/protein interfaces and may play an important role in the evolution of oligomeric proteins.

A Compact RNA Tertiary Structure Contains a Buried Backbone-K+ Complex

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Use of the rotation and translation functions

Publisher Summary The rotational search is carried out by looking for agreement between the Patterson functions of the search and target structures as a function of their relative orientation. In conventional programs the rotation function is calculated point by point on a grid spanning the asymmetric unit of angle space. For reasons of history and of compatibility with the translation function, most programs operate in reciprocal space. Once the orientation of a test molecule (and therefore of its symmetry mates) is known, the position of the molecule must be found. Many versions of a translational search have been proposed but the most commonly used is the T 1 function of Crowther and Blow. This function can also be interpreted in terms of the Patterson function or in reciprocal space. This survey of the most commonly used search procedures has attempted to clarify many practical matters in the application of the methods. A certain aura of mystery seems to surround search methods so that they are less frequently used than is perhaps appropriate.

Structure of deoxyhemoglobin a crystals grown from polyethylene glycol solutions

The structure of a new crystal form of deoxyhemoglobin A grown from polyethylene glycol solutions has been determined at 3·5 A resolution. The molecular orientations and positions were found by means of rotation and translation functions using the squared molecular transform of horse deoxyhemoglobin. Phases were calculated using atomic co-ordinates previously determined for deoxyhemoglobin A grown in another crystalline form. A difference Fourier synthesis showed minor structural differences near intermolecular contacts, the heme groups, and the β 1 carboxyl terminus. Some of these differences may be caused by the different crystalline environment; others may be due to errors in the analytical method. These apparent structural differences will be useful for interpreting results of a similar analysis of deoxyhemoglobin S crystals grown from the same solvent.