Douglas H. Juers

LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance.

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β‐galactosidase. The protein played a central role in Jacob and Monods development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino‐terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α‐complementation, in which addition to the inactive dimers of peptides containing the “missing” N‐terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X‐gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X‐ray structure represents an active conformation. Individual tetramers of β‐galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion‐like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

Cryo-cooling in macromolecular crystallography: advantages, disadvantages and optimization

The flash-cooling of crystals in macromolecular crystallography has become commonplace. The procedure makes it possible to collect data from much smaller specimens than was the case in the past Also, flash-cooled crystals are much less prone to radiation damage than their room-temperature counterparts, allowing data to be accumulated over extended periods of time. Notwithstanding the attractiveness of the technique, it does have potential disadvantages. First, better methods need to be developed to prevent damage to crystals on freezing. There is also a risk that structures determined at low temperature may suggest conclusions based on aspects of the structure that are not necessarily relevant at room temperature.

Inducing phase changes in crystals of macromolecules: Status and perspectives for controlled crystal dehydration

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device.

Gradual adaptive changes of a protein facing high salt concentrations.

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.

Progress in rational methods of cryoprotection in macromolecular crystallography.

Measurements of the average thermal contractions (294→72 K) of 26 different cryosolutions are presented and discussed in conjunction with other recent advances in the rational design of protocols for cryogenic cooling in macromolecular crystallography.

Direct and indirect roles of His‐418 in metal binding and in the activity of β‐galactosidase (E. coli)

The active site of ß‐galactosidase (E. coli) contains a Mg2+ ion ligated by Glu‐416, His‐418 and Glu‐461 plus three water molecules. A Na+ ion binds nearby. To better understand the role of the active site Mg2+ and its ligands, His‐418 was substituted with Asn, Glu and Phe. The Asn‐418 and Glu‐418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu‐418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg2+ site also reduce Na+ binding affinity. The Phe variant had reduced stability, bound Mg2+ weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His‐418, together with the Mg2+, modulate the central role of Glu‐461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His‐418 is very important for the formation of allolactose (the natural inducer of the lac operon).

The role of solvent transport in cryo-annealing of macromolecular crystals

Macromolecular crystals are usually cooled to approximately 100 K for X-ray diffraction experiments in order to diminish lattice damage arising from the ionizing radiation. Such cooling often produces lattice disorder, but this disorder can sometimes be substantially reduced by cycling the crystal between low and higher temperatures (called annealing). Here, two related aspects of cryocooling and annealing are investigated using crystals of beta-galactosidase and thermolysin. Firstly, as has been reported with other systems, there is an optimal cryoprotectant concentration above and below which diffraction is poor, with high mosaicity, diffuse scatter and low signal to noise. Measurements of the bulk density of the respective cryosolvents are consistent with the idea that at the optimal cryoprotectant concentration the contraction of the bulk solvent on cooling largely compensates for the contraction of the macromolecular lattice. Secondly, by controlling the relative humidity of the gas that contacts the crystal during the high (room) temperature phase, it is found that water is either imported into or exported out of the crystals during the melting phase of annealing. This water transport appears to change the concentration of the cryoprotectant solution and in so doing alters its thermal contraction. Thus, annealing appears to be involved, at least in part, in the tuning of the thermal contraction of the bulk solvent to best compensate for lattice contraction. Furthermore, it is found that if the cryoprotectant concentration is initially too high then annealing is more successful than if the concentration is initially too low. This result suggests that the search for optimal cryoprotectant conditions may be facilitated by equilibration of the crystal to relatively high cryoprotectant concentration followed by annealing.

The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography

Both crystallization and cryoprotection are often bottlenecks for high-resolution X-ray structure determination of macromolecules. Methylamine osmolytes are known stabilizers of protein structure. One such osmolyte, trimethylamine N-oxide (TMAO), has seen occasional use as an additive to improve macromolecular crystal quality and has recently been shown to be an effective cryoprotective agent for low-temperature data collection. Here, TMAO and the related osmolytes sarcosine and betaine are investigated as primary precipitating agents for protein crystal growth. Crystallization experiments were undertaken with 14 proteins. Using TMAO, seven proteins crystallized in a total of 13 crystal forms, including a new tetragonal crystal form of trypsin. The crystals diffracted well, and eight of the 13 crystal forms could be effectively cryocooled as grown with TMAO as an in situ cryoprotective agent. Sarcosine and betaine produced crystals of four and two of the 14 proteins, respectively. In addition to TMAO, sarcosine and betaine were effective post-crystallization cryoprotective agents for two different crystal forms of thermolysin. Precipitation reactions of TMAO with several transition-metal ions (Fe(3+), Co(2+), Cu(2+) and Zn(2+)) did not occur with sarcosine or betaine and were inhibited for TMAO at lower pH. Structures of proteins from TMAO-grown crystals and from crystals soaked in TMAO, sarcosine or betaine were determined, showing osmolyte binding in five of the 12 crystals tested. When an osmolyte was shown to bind, it did so near the protein surface, interacting with water molecules, side chains and backbone atoms, often at crystal contacts.

In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

The current version of the Cryobench in crystallo optical spectroscopy facility of the ESRF is presented. The diverse experiments that can be performed at the Cryobench are also reviewed.

Similarities and differences in radiation damage at 100 K versus 160 K in a crystal of thermolysin.

The temperature-dependence of radiation damage in macromolecular X-ray crystallography is currently much debated. Most protein crystallographic studies are based on data collected at 100 K. Data collection at temperatures below 100 K has been proposed to reduce radiation damage and above 100 K to be useful for kinetic crystallography that is aimed at the generation and trapping of protein intermediate states. Here the global and specific synchrotron-radiation sensitivity of crystalline thermolysin at 100 and 160 K are compared. Both types of damage are higher at 160 K than at 100 K. At 160 K more residue types are affected (Lys, Asp, Gln, Pro, Thr, Met, Asn) than at 100 K (Met, Asp, Glu, Lys). The X-ray-induced relative atomic B-factor increase is shown to correlate with the proximity of the atom to the nearest solvent channel at 160 K. Two models may explain the observed correlation: either an increase in static disorder or an increased attack of hydroxyl radicals from the solvent area of the crystal.