Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass Spectrometry

The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (·OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as + 16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by ·OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen–deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.     

The Cleaved N-Terminus of pVI Binds Peripentonal Hexons in Mature Adenovirus

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.     

Quantitative analysis of glycation and its impact on antigen binding

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.     

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes

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Chromophore Packing Leads to Hysteresis in GFP

Green fluorescent protein (GFP) possesses a unique folding landscape with a dual basin leading to the hysteretic folding behavior observed in experiment. While theoretical data do not have the resolution necessary to observe details of the chromophore during refolding, experimental results point to the chromophore as the cause of the observed hysteresis. With the use of NMR spectroscopy, which probes at the level of the individual residue, the hysteretic intermediate state is further characterized in the context of the loosely folded isomerized native-like state {N_iso} predicted in simulation. In the present study, several residues located in the lid of GFP indicate heterogeneity of the native states. Some of these residues show chemical shifts when the native-like intermediate {N_iso} responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in {N_iso} as predicted in recent theoretical work. Here, we observed that multiple chromophore environments within the native state are averaged in the trapped intermediate, linking chromophore flexibility to mispacking in the trapped intermediate. The present work is experimental evidence for the proposed final “locking” mechanism in GFP folding forming an incorrectly or loosely packed barrel under intermediate (hysteretic) folding conditions.