Water-mediated Transformation Research Articles

Front Cover: Water‐Mediated Structural Transformations of CuII 5‐Halonicotinates Coordination Networks with Distinct Mechanisms (Chem. Eur. J. 53/2017)

Front Cover: Water‐Mediated Structural Transformations of Cu^II 5‐Halonicotinates Coordination Networks with Distinct Mechanisms (Chem. Eur. J. 53/2017)

Anion-Induced Formation of Lanthanide-Organic Chains From 3D Framework Solids. Anion Exchange in a Crystal-to-Crystal Manner

A water-mediated transformation reversibly converts the neutral 3D microporous coordination polymers [Ln2(adipate)3(H2O)4]·6H2O (Ln3+ = Pr3+) and [Ln2(adipate)3(H2O)4]xH2O (Ln3+ = Gd3+, Sm3+), into the ionic 1D polymer [Ln(adipate)(H2O)5]XH2O (with X = NO3−, Cl− and Ln3+ = Pr3+, Gd3+, Sm3+). The 1D polymer consists of positively charged hydrogen-bonded lanthanide−adipate chains enclathrating NO3− or Cl− anions acting as templates. The hydrogen bonds linking the chains between them are so strong that it is possible to exchange NO3− for Cl− and vice versa in a crystal-to-crystal manner. The anion exchange reactions were directly observed on crystals by using Raman microprobe spectroscopy, single-crystal X-ray diffraction, and scanning electron microscopy.

Water-mediated variability in the structure of relaxed-state haemoglobin.

The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h. 79%, the water content of the crystal is reduced and the molecule appears to move towards the R2 state. The crystals undergo a water-mediated transformation involving a doubling of one of the unit-cell parameters and an increase in water content when the environmental humidity is further reduced to r.h. 75%. The water content is now similar to that in the native crystals and the molecules are in the R state. The crystal structure at r.h. 66% is similar, but not identical, to that at r.h. 75%, but the solvent content is substantially reduced and the molecules have a quaternary structure that is in between those corresponding to the R and R2 states. Thus, variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action.

An asymmetric dimer of β‐lactoglobulin in a low humidity crystal form—Structural changes that accompany partial dehydration and protein action

Dimeric lactoglobulin molecules exist in the open conformation at basic pH, whereas they exist in the closed conformation at acidic pH, after undergoing the Tanford transition around neutral pH. Orthorhombic crystals consisting of molecules in the open conformation, grown close to neutral pH, undergo a water-mediated transformation when the relative humidity around the crystals is reduced. The two subunits in the dimer are related by a crystallographic twofold axis in the native crystals while the dimer is asymmetric in the low humidity form. Interestingly, one of the subunits in the dimer in the low humidity form is in an open conformation while the other is in a closed conformation. This is the first observation of such an asymmetric dimer. A hydrogen bond between the side chains of Gln35 and Tyr42 exists and the side chain of Glu89 is substantially buried in the closed subunit of the asymmetric unit, as in other structures with molecules in the closed conformation. However, the closure of the EF loop is not complete; its conformation can be described as half-closed. A comparison of different crystal structures of beta-lactoglobulin indicates that the conformation of the loops in the molecule is substantially influenced by other factors such as crystal packing, the pH, and the composition of the medium, while the change in the conformation of the EF loop follows the Tanford transition. The mutual disposition of the two subunits in the low humidity form is halfway between those in the open and closed structures. The present work further demonstrates that structural changes that occur during partial dehydration could mimic those that occur during the action of proteins.

Structures of human oxy- and deoxyhaemoglobin at different levels of humidity: variability in the T state.

High-salt crystals of human oxy- and deoxyhaemoglobin have been studied at different levels of environmental humidity and solvent content. The structure of the oxy form remains relatively unchanged at all levels. The deoxy form, however, undergoes a water-mediated transformation when the relative humidity around the crystals is reduced below 93%. The space group is maintained during the transformation, but the unit-cell volume nearly doubles, with two tetrameric molecules in the asymmetric unit of the low-humidity form compared with one in the native crystals. Interestingly, the haem geometry in the low-humidity form is closer to that in the oxy form than to that in the native deoxy form. The quaternary structure of one of the tetramers moves slightly towards that in the oxy form, while that in the other is more different from the oxy form than that in the high-salt native deoxy form. Thus, it would appear that, as in the case of the liganded form, the deoxy form of haemoglobin can also access an ensemble of related T states.

Role of water in plasticity, stability, and action of proteins: the crystal structures of lysozyme at very low levels of hydration.

Earlier studies involving water-mediated transformations in lysozyme and ribonuclease A have shown that the overall movements in the protein molecule consequent to the reduction in the amount of surrounding water are similar to those that occur during enzyme action, thus highlighting the relationship among hydration, plasticity, and action of these enzymes. Monoclinic lysozyme retains its crystallinity even when the level of hydration is reduced further below that necessary for activity (about 0.2 gram of water per gram of protein). In order to gain insights into the role of water in the stability and the plasticity of the protein molecule and the geometrical basis for the loss of activity that accompanies dehydration, the crystal structures of monoclinic lysozyme with solvent contents of 17.6%, 16.9%, and 9.4% were determined and refined. A detailed comparison of these forms with the normally hydrated forms show that the C-terminal segment (residues 88-129) of domain I and the main loop (residues 65-73) in domain II exhibit large deviations in atomic positions when the solvent content is reduced, although the three-dimensional structure is essentially preserved. Many crucial water bridges between different regions of the molecule are conserved in spite of differences in detail, even when the level of hydration is reduced well below that required for activity. The loss of activity that accompany dehydration appears to be caused by the removal of functionally important water molecules from the active-site region and the reduction in the size of the substrate binding cleft.

High-Resolution Crystals of Methionine Aminopeptidase fromPyrococcus furiosusObtained by Water-Mediated Transformation

The monoclinic crystal form of methionine aminopeptidase fromPyrococcus furiosus(MAP-Pfu) has been crystallized from four different conditions. Native crystals belong to space group P21with typical unit-cell dimensionsa= 53.4,b= 85.1,c= 72.7 Å, β = 107.7° and diffract to 2.9–4.5 Å resolution. However, there is a problem of nonisomorphism among the crystals. Water-mediated transformation to low-humidity form occurs by reduction of the relative humidity of crystal environment to 79%. The unit-cell dimensions of transformed crystals area= 51.9,b= 83.3,c= 70.3 Å, β = 105.9°, and the calculated solvent content is 3.9% less than in original crystals. Transformation to low-humidity form is accompanied by 1.7 times reduction of overall temperature factors, extension of diffraction resolution up to 1.75 Å, without change or reduction of crystal mosaicity, and improvement in stability to X-ray radiation. The water-mediated transformation also appears to relieve the problem of nonisomorphism among the original MAP-Pfu crystals.

An X-ray analysis of native monoclinic lysozyme. A case study on the reliability of refined protein structures and a comparison with the low-humidity form in relation to mobility and enzyme action.

The atomic models of native monoclinic lysozyme obtained by refinement at Bangalore and elsewhere [Young, Dewan, Nave & Tilton (1993). J. Appl. Cryst. 26, 309-319] differed significantly in the flexible regions of the protein molecule. The two models were reconciled starting from regions where they were in reasonable agreement to produce an improved model which yielded an R value of 0.169 for 12 816 observed reflections in the 10-2 A resolution range. The reconciled model was compared with the structure of the 88% relative humidity form obtained through a water-mediated transformation [Madhusudan, Kodandapani & Vijayan (1993). Acta Cryst. D49, 234-245]. Parts of the flexible regions of the molecule register significant movements during the transformation. The changes resulting from the transformation from the native to the low-humidity forms are pronounced in many of the side chains in the active-site region, thus indicating the relationship between hydration, mobility and enzyme action. The fact that the overall changes in molecular geometry resulting from water-mediated transformation are similar to those which occur during enzyme action, further emphasizes this relationship.

Characterization of lysozyme crystals with unusually low solvent content

Studies on the low-humidity (88%) forms of tetragonal and monoclinic lysozyme, resulting from water-mediated transformations, have provided a wealth of information on the variability in protein hydration, its structural consequences and the water structure associated with proteins, in addition to facilitating the delineation of the rigid and the flexible regions in the protein molecule and the invariant features in its hydration shell. Surprisingly, monoclinic lysozyme continues to diffract even when the environmental humidity is drastically reduced, thus permitting the structural study of the enzyme at different levels of hydration. As part of a study in this direction, three very low humidity forms, two of them occurring at a nominal relative humidity of 38% and the other at 5% relative humidity, have been characterized. These have unprecedented low solvent contents of 16.9, 17.6 and 9.4%, respectively, as determined by the Matthews method.

Water-dependent domain motion and flexibility in ribonuclease A and the invariant features in its hydration shell. An X-ray study of two low-humidity crystal forms of the enzyme.

The crystal structures of 88 and 79% relative humidity forms of ribonuclease A, resulting from water-mediated transformations, have been refined employing the restrained least-squares method using X-ray data collected on an area detector to R = 0.173 for 15 326 observed reflections in the 10-1.5 A resolution shell and R = 0.176 for 8534 observed reflections in the 10-1.8 A shell, respectively. The comparison of these structures with those of the native, the phosphate-bound and the sulfate-bound forms demonstrates that the mobility of the ribonuclease A molecule involves hinge-bending movement of the two domains and local flexibility within them, particularly at the termini of regular secondary structures and in loops. The comparison also leads to the identification of 31 invariant water molecules in the hydration shell of the enzyme, many of which are involved in holding different parts of the molecule together and in stabilizing local structure. The conformational changes that accompany the partial removal of the surrounding water, particularly those observed in the 79% form, could be similar to those that occur during enzyme action.

Protein hydration and water structure: X-ray analysis of a closely packed protein crystal with very low solvent content.

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10-1.75 A resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect beta-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding.

Water-mediated transformations in protein crystals

Different crystal forms of bovine pancreatic ribonuclease A and hen egg white lysozyme, 2Zn insulin, 4Zn insulin and crystals of concanavalin A were examined under controlled environmental humidity in the relative humidity (r.h.) range of 100 to 75%. Many of them, but not all, undergo reversible structural transformations as evidenced by discontinuous changes in the diffraction pattern, the unit-cell dimensions and the solvent content. Tetragonal, orthorhombic and monoclinic lysozyme and a new crystal form of ribonuclease A show transformations at r.h.'s above 90%. Monoclinic lysozyme transforms at low r.h. to another monoclinic form with nearly half the original cell volume. The well known monoclinic form of ribonuclease A grown from aqueous ethanol solution undergoes two transformations while the same form grown from 2-methyl-2,4pentanediol (MPD) solution in phosphate buffer does not transform at all. Soaking experiments involving alcohol solutions demonstrate that MPD has the effect of decreasing the r.h. at which the transformation occurs. Triclinic lysozyme, 2Zn insulin, 4Zn insulin and the crystals of cancanavalin A do not transform in the 100 to 75% r.h. range before losing crystallinity. The results obtained so far indicate that the crystal structure has a definite influence on water-mediated transformations. The transformations do not appear to depend critically on the amount of solvent in the crystals but the r.h. at which they occur is influenced by the composition of the solvent. The transformations appear to involve changes in crystal packing as well as conformational transitions in protein molecules. The present investigations and other related studies suggest that water-mediated transformations in protein crystals could be very useful in

Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.

Environmental effects of water-mediated transformations in the crystals of ribonuclease A

Environmental effects of water-mediated transformations in the crystals of ribonuclease A