Membrane Protein Structural Validation by Oriented Sample Solid-State NMR: Diacylglycerol Kinase

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.     

Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel

Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.     

Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus

Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.     

Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers

The interactions of ¹⁵N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of ³¹P chemical shielding and ¹⁴N quadrupolar interactions and by the MD simulation.     

Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å

The dibenzothiophene (DBT) monooxygenase DszC, which is the key initiating enzyme in “4S” metabolic pathway, catalyzes sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO₂). Here, we report the crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Intriguingly, two distinct conformations occur in the flexible lid loops adjacent to the active site (residue 280–295, between α9 and α10). They are named “open”' and “closed” state respectively, and might show the status of the free and ligand‐bound DszC. The molecular docking results suggest that the reduced FMN reacts with an oxygen molecule at C4a position of the isoalloxazine ring, producing the C4a‐(hydro)peroxyflavin intermediate which is stabilized by H391 and S163. H391 may contribute to the formation of the C4a‐(hydro)peroxyflavin by acting as a proton donor to the proximal peroxy oxygen, and it might also be involved in the protonation process of the C4a‐(hydro)xyflavin. Site‐directed mutagenesis study shows that mutations in the residues involved either in catalysis or in flavin or substrate‐binding result in a complete loss of enzyme activity, suggesting that the accurate positions of flavin and substrate are crucial for the enzyme activity. Proteins 2014; 82:1708–1720. © 2014 Wiley Periodicals, Inc.     

Soft interactions and crowding

The intracellular milieu is complex, heterogeneous and crowded—an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.     

过氧化氢酶-无机杂化纳米花的制备及催化特性

过氧化氢酶(catalase,CAT)是一种在食品、医疗、纺织等领域广泛应用的工业酶,具有催化效率高、专一性强、绿色环保等突出特点。工业中游离过氧化氢酶无法回收再利用,导致以其为核心的工业生物转化过程成本较高。开发一种简单、温和、低成本并且体现绿色化学理念的方法对过氧化氢酶进行固定化有望提高其利用率并且强化酶学性能,具有迫切的现实需求。本研究将源自枯草芽孢杆菌(Bacillus subtilis)168的过氧化氢酶KatA在大肠杆菌中进行重组表达,之后将分离纯化得到的纯酶以酶-无机杂化纳米花形式制备成固定化酶并进行酶学性质研究。结果显示,利用乙醇沉淀、DEAE阴离子交换层析、疏水层析3步纯化,最终获得电泳纯的重组KatA,之后通过优化制备条件获得了一种新型KatA/Ca₃(PO₄)₂杂化纳米花固定化酶。酶学性质研究结果显示,游离酶KatA的最适反应温度为35℃,KatA/Ca₃(PO₄)₂杂化纳米花的最适反应温度为30−35℃,二者最适反应pH值均为11.0。游离酶KatA和KatA/Ca₃(PO₄)₂杂化纳米花在pH4.0−11.0和25−50℃条件下均表现出较好的稳定性。KatA/Ca₃(PO₄)₂杂化纳米花显示出比游离酶KatA更好的储存稳定性,在4℃储存14d后仍保留82%的酶活力,而游离酶仅具有50%的酶活力。此外,纳米花在进行5次催化反应后仍具有55%的酶活力,表明其具有较好的操作稳定性。动力学研究结果显示,游离酶KatA对底物过氧化氢的K_m为(8.80±0.42)mmol/L,k_cat/K_m为(13151.53±299.19)L/(mmol·s);而KatA/Ca₃(PO₄)₂杂化纳米花的K_m为(32.75±2.96)mmol/L,k_cat/K_m为(4550.67±107.51)L/(mmol·s)。与游离酶KatA相比,KatA/Ca₃(PO₄)₂杂化纳米花对底物过氧化氢的亲和力下降,同时其催化效率也有所降低。综上所述,本研究以Ca²⁺作为自组装诱导剂,成功将KatA以酶-无机杂化纳米花形式制备成固定化酶,不仅对部分酶学性能实现了强化,而且为固定化过氧化氢酶的绿色制备和规模化应用奠定了基础。