Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling.

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/µL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.

The peripheral neuropathy-linked Trembler and Trembler-J mutant forms of peripheral myelin protein 22 are folding-destabilized.

Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D ( Trembler phenotype) and L16P ( Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the three-dimensional structures of these disease-linked mutants are similar to that of the folded wild-type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild-type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild-type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild-type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant.

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

Variation in FPOP Measurements Is Primarily Caused by Poor Peptide Signal Intensity.

Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions. Graphical Abstract ᅟ.

Optimal mutation sites for PRE data collection and membrane protein structure prediction.

Nuclear magnetic resonance paramagnetic relaxation enhancement (PRE) measures long-range distances to isotopically labeled residues, providing useful constraints for protein structure prediction. The method usually requires labor-intensive conjugation of nitroxide labels to multiple locations on the protein, one at a time. Here a computational procedure, based on protein sequence and simple secondary structure models, is presented to facilitate optimal placement of a minimum number of labels needed to determine the correct topology of a helical transmembrane protein. Tests on DsbB (four helices) using just one label lead to correct topology predictions in four of five cases, with the predicted structures <6 Å to the native structure. Benchmark results using simulated PRE data show that we can generally predict the correct topology for five and six to seven helices using two and three labels, respectively, with an average success rate of 76% and structures of similar precision. The results show promise in facilitating experimentally constrained structure prediction of membrane proteins.

Purification and initiation of structural characterization of human peripheral myelin protein 22, an integral membrane protein linked to peripheral neuropathies.

Gene duplications, deletions, and point mutations in peripheral myelin protein 22 (PMP22) are linked to several inherited peripheral neuropathies. However, the structural and biochemical properties of this very hydrophobic putative tetraspan integral membrane protein have received little attention, in part because of difficulties in obtaining milligram quantities of wild type and disease-linked mutant forms of the protein. In this study a fusion protein was constructed consisting of a fragment of lambda repressor, a decahistidine tag, an intervening TEV protease cleavage site, a Strep tag, and the human PMP22 sequence. This fusion protein was expressed in Escherichia coli at a level of 10-20 mg/L of protein. Following TEV cleavage of the fusion partner, PMP22 was purified and its structural properties were examined in several different types of detergent micelles using cross-linking, near and far-UV circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy. PMP22 is highly helical and, in certain detergents, shows evidence of stable tertiary structure. The protein exhibits a strong tendency to dimerize. The 1H-15N TROSY NMR spectrum is well dispersed and contains signals from all regions of the protein. It appears that detergent-solubilized PMP22 is amenable to detailed structural characterization via crystallography or NMR. This work sets the stage for more detailed studies of the structure, folding, and misfolding of wild type and disease-linked mutants in order to unravel the molecular defects underlying peripheral neuropathies.

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.

Metal ion scrambling in hexanuclear M(6)(Et(2)NCO(2))(12) complexes (M = Co, Mg). Synthesis, solid state structure, and solution dynamics of heteronuclear Co(n)Mg(6-n)(Et(2)NCO(2))(12) complexes.

Heteronuclear diethylcarbamato complexes of the form Co(n)()Mg(6)(-)(n)()(Et(2)NCO(2))(12) were prepared from the isostructural homonuclear precursors Mg(6)(Et(2)NCO(2))(12), 1, and Co(6)(Et(2)NCO(2))(12), 2, via a solvothermal methodology. Two materials were selected for single-crystal X-ray diffraction analysis: Co(1.6)Mg(4.4)(Et(2)NCO(2))(12) and Co(2.7)Mg(3.3)(Et(2)NCO(2))(12). Both compounds crystallize in the orthorhombic space group Ccca, as do 1 and 2. The molecular structure is best described as two trinuclear M(3) units cross-linked by diethylcarbamate ligands and twisted about one another, so that the complex has overall D(2) symmetry and is chiral. Each trinuclear unit consists of two terminal pentacoordinate metal ions and one central hexacoordinate metal ion. The X-ray diffraction data were unambiguous that the Co(2+) ions migrate exclusively to the pentacoordinate sites in the heteronuclear complexes, thus demonstrating that metal ion scrambling at the molecular level must occur. The composition of individual crystals can be continuously varied for Co(2+) mole fractions chi(Co) < 0.5, and the a and c unit cell distances are linearly related to chi(Co). This indicates that the compounds behave as solid solutions. There appears to be either a chemical or crystallographic phenomenon inherent in the synthetic methodology that prevents isolation of heteronuclear materials having chi(Co) > 0.5. Solution electronic spectroscopy and molecular weight measurements show that 2 can dissociate in chloroform and cyclohexane solution to give a dimeric complex 2'. This behavior contrasts with the stability of 1 in solution, as shown by NMR. The kinetic rate profile for formation of Co(n)Mg(6-n)(Et(2)NCO(2))(12) reveals saturation kinetics and is consistent with direct attack by 2' on 1 to give the heteronuclear complex via a higher nuclearity intermediate. This study illustrates a general method for the preparation of solids based on heteronuclear Werner-type complexes of the M(6)(Et(2)NCO(2))(12) structure type, and the mechanism by which such compounds can be formed from isostructural homonuclear precursors.

EPR and magnetic properties of heteronuclear Mn(n)Mg(6-)(n)(O(2)CNEt(2))(12): impact of structural distortions on Mn(II) in weak ligand fields.

The reaction between Mn(6)L(12) and Mg(6)L(12) (L = N,N-diethylcarbamate) results in isolation of heteronuclear complexes Mn(n)Mg(6)(-)(n)L(12). A series was prepared with different doping factors n by varying the Mn/Mg ratio in the crystallization solutions. Single-crystal X-ray diffraction shows that MnMg(5)L(12) is isostructural with Mn(6)L(12) and Mg(6)L(12). Magnetic susceptibility data on the series Mn(n)Mg(6)(-)(n)L(12) (n = 1-6) are consistent with antiferromagnetic Mn.Mn interactions. At low n, the magnetic data demonstrate the formation of magnetically isolated Mn(2+) centers. This was confirmed by measurement of the EPR spectrum at a doping factor n = 0.06 in solution, as a powder, and as single crystals. These show hyperfine interactions consistent with isolated Mn(2+). The EPR spectrum of Mn(0.06)Mg(5.94)L(12) exhibits a dominant signal at g(eff) = 4, and a wide series of less intense signals spanning 200-6000 G in the X-band regime. This unusual behavior in a weak-field Mn(2+) complex is attributed to the substantial distortions from cubic ligand field geometry in this system. The g(eff) = 4 signals are attributed to a C(2)-symmetric hexacoordinate Mn(2+) ion with D > 0.3 cm(-)(1) and E/D = 0.33. The wide series is assigned to an axial C(4)(v) pentacoordinate Mn(2+) site with D = 0.05 cm(-)(1). Comparison of the g(eff) = 4 signals to the g = 4.1 signals exhibited by the tetramanganese complex in photosystem II belies the fact that they almost certainly arise from different spin systems. In addition, the similarity of the spectrum of Mn(n)Mg(6)(-)(n)L(12) to mononuclear Mn(4+) complexes suggests that considerable care must be exercised in the use of EPR as a fingerprint for the manganese oxidation state, particularly in manganese proteins where molecular composition may not be precisely established.