Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases.

Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

Role of lysine during protein modification by HOCl and HOBr: halogen-transfer agent or sacrificial antioxidant?

Although protein degradation by neutrophil-derived hypochlorous acid (HOCl) and eosinophil-derived hypobromous acid (HOBr) can contribute to the inactivation of pathogens, collateral damage to host proteins can also occur and has been associated with inflammatory diseases ranging from arthritis to atherosclerosis. Though previous research suggested halotyrosines as biomarkers of protein damage and lysine as a mediator of the transfer of a halogen to tyrosine, these reactions within whole proteins are poorly understood. Herein, reactions of HOCl and HOBr with three well-characterized proteins [adenylate kinase (ADK), ribose binding protein, and bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the proteins: (1) covalent modification of electron-rich amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of protein backbones (via sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and HOBr converted lysine into lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform oxidant levels, fragmentation and loss of secondary structure correlated with protein size. To further examine the role of lysine, a lysine-free ADK variant was rationally designed. The absence of lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that lysine serves predominantly as a sacrificial antioxidant (via formation of lysine nitrile) toward HOCl and as a halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with HOBr.

'q-Titration' of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy.

'q-Titration' refers to the systematic comparison of signal intensities in solution NMR spectra of uniformly (15)N labeled membrane proteins solubilized in micelles and isotropic bicelles as a function of the molar ratios (q) of the long-chain lipids (typically DMPC) to short-chain lipids (typically DHPC). In general, as q increases, the protein resonances broaden and correspondingly have reduced intensities due to the overall slowing of protein reorientation. Since the protein backbone signals do not broaden uniformly, the differences in line widths (and intensities) enable the narrower (more intense) signals associated with mobile residues to be differentiated from the broader (less intense) signals associated with "structured" residues. For membrane proteins with between one and seven trans-membrane helices in isotropic bicelles, we have been able to find a value of q between 0.1 and 1.0 where only signals from mobile residues are observed in the spectra. The signals from the structured residues are broadened so much that they cannot be observed under standard solution NMR conditions. This q value corresponds to the ratio of DMPC:DHPC where the signals from the structured residues are "titrated out" of the spectrum. This q value is unique for each protein. In magnetically aligned bilayers (q>2.5) no signals are observed in solution NMR spectra of membrane proteins because the polypeptides are "immobilized" by their interactions with the phospholipid bilayers on the relevant NMR timescale (∼10(5)Hz). No signals are observed from proteins in liposomes (only long-chain lipids) either. We show that it is feasible to obtain complementary solution NMR and solid-state NMR spectra of the same membrane protein, where signals from the mobile residues are present in the solution NMR spectra, and signals from the structured residues are present in the solid-state NMR spectra. With assigned backbone amide resonances, these data are sufficient to describe major features of the secondary structure and basic topology of the protein. Even in the absence of assignments, this information can be used to help establish optimal experimental conditions.

CHOBIMALT: a cholesterol-based detergent.

Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3−4 µM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1.

Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts.

A method for assigning solid-state NMR spectra of membrane proteins aligned in phospholipid bicelles that makes use of isotropic chemical shift frequencies and assignments is demonstrated. The resonance assignments are based on comparisons of 15N chemical shift differences in spectra obtained from samples with their bilayer normals aligned perpendicular and parallel to the direction of the applied magnetic field.

Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy.

The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively (15)N-labeled samples to resolve and assign the backbone amide resonances and to measure the associated (15)N chemical shift and (1)H-(15)N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. (1)H/(13)C/(15)N triple-resonance experiments were applied to selectively (13)C'- and (15)N-labeled samples to complete the resonance assignments, especially for residues in the nonhelical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers.

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.

SPINAL modulated decoupling in high field double- and triple-resonance solid-state NMR experiments on stationary samples.

Continuous wave irradiation has limited bandwidth for heteronuclear 1H decoupling at high fields and for 13C decoupling in 1H/13C/15N triple-resonance experiments. SPINAL-16 modulation is shown to improve the efficiency of 1H and 13C heteronuclear decoupling on single crystals of peptides and on magnetically aligned samples of membrane proteins in bicelles, which is of particular importance because aqueous samples of biomolecules are lossy at high fields, which limits the strengths of the RF fields that can be applied.

NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system.

The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone (1)H-(15)N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the (1)H-(15)N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The (1)H-(15)N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 A. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.

High-resolution NMR spectroscopy of membrane proteins in aligned bicelles.

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientational information necessary for protein structure determination.