Sol M. Gruner

Entrapment of carbon dioxide in the active site of carbonic anhydrase II

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

A short, strong hydrogen bond in the active site of human carbonic anhydrase II.

The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 A resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 A. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO(2) in the enzyme-substrate complex, a weak binding that enhances k(cat) for the conversion of CO(2) into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pK(a) of the zinc-bound water and to promote proton transfer in catalysis.

High-pressure cooling of protein crystals without cryoprotectants.

Flash-cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and in some cases unsuccessful. A method is described to cool protein crystals in high-pressure helium gas without the need for penetrative cryoprotectants. The method involves mounting protein crystals from the native mother liquor in a cryoloop with a droplet of oil, pressurizing the crystal to 200u2005MPa in He gas, cooling the crystal under pressure and then releasing the pressure. The crystal is then removed from the apparatus under liquid nitrogen and handled thereafter like a normal cryocooled crystal. Results are presented from three representative proteins. Dramatic improvement in diffraction quality in terms of resolution and mosaicity was observed in all cases. A mechanism for the pressure cooling is proposed involving high-density amorphous (HDA) ice which is produced at high pressure and is metastable at room pressure and 110u2005K.

The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring.

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.

Evidence for liquid water during the high-density to low-density amorphous ice transition

Polymorphism of water has been extensively studied, but controversy still exists over the phase transition between high-density amorphous (HDA) and low-density amorphous (LDA) ice. We report the phase behavior of HDA ice inside high-pressure cryocooled protein crystals. Using X-ray diffraction, we demonstrate that the intermediate states in the temperature range from 80 to 170 K can be reconstructed as a linear combination of HDA and LDA ice, suggesting a first-order transition. We found evidence for a liquid state of water during the ice transition based on the protein crystallographic data. These observations open the possibility that the HDA ice induced by high-pressure cryocooling is a genuine glassy form of high-density liquid.

Solution of protein crystallographic structures by high-pressure cryocooling and noble-gas phasing

Room-pressure flash-cryocooling of protein crystals is the standard way to reduce radiation damage during data collection. Typically, it is necessary to find cryoprotection conditions by trial and error, a process that is not always successful. Recently, a new method, high-pressure cryocooling, was developed that does not require penetrative cryoprotectants and typically yields very high quality diffraction. Since this method involves helium gas as a pressurizing medium, it was of great interest to see whether the method could be extended to diffraction phasing by the incorporation of heavy noble gases such as krypton. A modified Kr-He high-pressure cyrocooling procedure is described wherein crystals are first pressurized with krypton gas to 10 MPa for 1 h. The krypton pressure is then released and the crystals are repressurized with helium over 150 MPa and cooled to liquid-nitrogen temperatures. Porcine pancreas elastase (PPE; 240 residues, 26 kDa) was selected as a test case for this study. Excellent diffraction was achieved by high-pressure cryocooling without penetrating cryoprotectants. A single 0.31 occupied krypton site in a PPE molecule [Bijvoet amplitude ratio (|DeltaF|/F) of 0.53%] was successfully used for SAD phasing at 1.3 A. This method has the potential to greatly simplify obtaining protein structures.

Protein dynamical transition at 110 K

Proteins are known to undergo a dynamical transition at around 200 K but the underlying mechanism, physical origin, and relationship to water are controversial. Here we report an observation of a protein dynamical transition as low as 110 K. This unexpected protein dynamical transition precisely correlated with the cryogenic phase transition of water from a high-density amorphous to a low-density amorphous state. The results suggest that the cryogenic protein dynamical transition might be directly related to the two liquid forms of water proposed at cryogenic temperatures.

High-pressure cryocooling for capillary sample cryoprotection and diffraction phasing at long wavelengths.

Crystal cryocooling is usually employed to reduce radiation damage during X-ray crystallography. Recently, a high-pressure cryocooling method has been developed which results in excellent diffraction-quality crystals without the use of penetrative cryoprotectants. Three new developments of the method are presented here: (i) Xe-He high-pressure cryocooling for Xe SAD phasing, (ii) native sulfur SAD phasing and (iii) successful cryopreservation of crystals in thick-walled capillaries without additional cryoprotectants other than the native mother liquor. These developments may be useful for structural solution of proteins without the need for selenomethionine incorporation and for high-throughput protein crystallography.

Generation dependent mesophase behavior in extended amphiphilic dendrons in the shape of macromolecular dumbbells.

Small angle X-ray scattering studies of 2nd and 3rd generation based extended amphiphilic dendrons in the shape of macromolecular dumbbells with identical hydrophilic volume fractions suggest 2-D hexagonal columnar and Pm3n micellar cubic mesophases, respectively, elucidating the role of shape induced interface curvature in mesophase formation.

High pressure cooling of protein crystals without cryoprotectants

Flash-cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and in some cases unsuccessful. A method is described to cool protein crystals in high-pressure helium gas without the need for penetrative cryoprotectants. The method involves mounting protein crystals from the native mother liquor in a cryoloop with a droplet of oil, pressurizing the crystal to 200 MPa in He gas, cooling the crystal under pressure and then releasing the pressure. The crystal is then removed from the apparatus under liquid nitrogen and handled thereafter like a normal cryocooled crystal. Results are presented from three representative proteins. Dramatic improvement in diffraction quality in terms of resolution and mosaicity was observed in all cases. A mechanism for the pressure cooling is proposed involving high-density amorphous (HDA) ice which is produced at high pressure and is metastable at room pressure and 110 K.