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The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

TGF-β1, TGF- β2, and TGF-β3 are 26 kDa disulfide-linked homodimeric signaling proteins. They all signal through the TGF-β type I and type II receptors, yet TGF-β2, which is well known to bind TβRII several-hundred fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for the TGF-β type III receptor (TβRIII), a membrane-anchored non-signaling receptor that potentiates the binding of TβRII. Though it is known that TβRIII has two component domains that bind TGF-β2 non-cooperatively at independent sites, the structure of these domains bound to TGF-β2 and residues responsible for specific binding are not yet known.

Cytochrome c (cyt-c) plays a key role in activating intrinsic apoptosis, a closely regulated cell death mechanism. Complete understanding of this process has implications for treatment of neurodegenerative diseases like Huntington's disease, as well as cancer. A pivotal signaling event in this apoptotic pathway is the peroxidation of the mitochondrial lipid cardiolipin (CL) by cyt-c. Cyt-c gains CL-specific lipid peroxidase activity by binding to CL during apoptosis, but exactly what structural changes lead to its proapoptotic activity have remained elusive. We have performed both structural and functional measurements to determine changes in structure and dynamics of cyt-c that accompany the activation of its peroxidase function upon binding to CL. Specifically, we used FTIR and multidimensional magic-angle-spinning (MAS) solid-state NMR (ssNMR) on samples mimicking proapoptotic conditions that trigger cyt-c's peroxidase activity. Comparison to FTIR and solution NMR data for the soluble protein revealed that cyt-c did not experience major unfolding upon membrane binding. We probed the protein-lipid interactions via ssNMR on both the protein and the lipids, and additionally used fluorescence quenching measurements of resonance energy transfer between the cyt-c heme and fluorescently labeled lipids. We conclude that increases in peroxidase activity of peripherally CL-bound cyt-c do not result from extensive unfolding of cyt-c. We instead propose an alternative mechanism in which localized structural changes, rather than global unfolding, in the CL-bound protein may be enough to allow for significant lipid peroxidation function to arise.

More than 20 human diseases are associated with amyloid fibril formation, one of which is Huntington's Disease (HD). HD, which poses a risk for ∼200,000 Americans, is a neurodegenerative disease that degrades cognitive and motor functions and is one of ten disorders to feature polyglutamine (polyQ) domain expansion. The self-aggregation of expanded polyQ segments in the huntingtin protein (htt) leads to fibril formation. Flanking regions of the polyQ domain affect the kinetics of this aggregation. As protein aggregation appears to correlate to disease onset, we aim to understand the structure of the mature fibrils, a critical step toward resolving structural changes along the fibril formation pathway.Fibrils of site-specific isotopically labeled peptides mimicking polyQ-containing N-terminal fragments of the htt protein were studied by magic-angle-spinning (MAS) solid-state NMR (ssNMR). We probed the conformation of both the polyQ amyloid core and its flanking regions by multidimensional MAS ssNMR, finding an a-helical conformation for residues in the very N-terminus (httNT), which is known to initiate the aggregation process. Structural models of the aggregation pathway and fibril were assessed based on structural data, relaxation times, and order parameters from MAS ssNMR measurements. Fibrils featuring mutations in the linker region connecting the httNT helix to the amyloid core were also investigated. Earlier animal studies have shown these mutations to reduce toxicity, and EM shows significant changes in fibril morphology. The site-specific effects of these and other mutations, on both the httNT and the polyQ core, were explored through structural and dynamics measurements. The characterization of the mutants, which mimic post-translational Ser phosphorylation, suggests biophysical connections between the stability and molecular structure of species along the aggregation pathway and the observed changes in toxicity.

The recognition and binding of specific lipid species is critical for the cellular targeting, folding and activity of various membrane-associated or peripherally bound proteins. These interactions facilitate localization of the protein in question in the proper membrane in the cell. In certain cases, binding to cholesterol is thought to play a key role in the targeting of membrane-associated proteins to cholesterol-rich membranes, and to laterally-segregated cholesterol-enriched domains within the plasma membrane. Previous studies have identified a cholesterol-binding motif that may be found in numerous proteins. This relatively short cholesterol recognition/interaction amino acid consensus (CRAC) is thought to enable tight cholesterol binding, both in a protein-context and in isolation. Experimental challenges have limited the availability of structural data on the molecular or biophysical nature of this interaction. Using solid-state NMR (ssNMR), we explore the structural features of this domain as found in the cholesterol-binding protein caveolin 1. The use of ssNMR permits the study of this cholesterol-binding domain in cholesterol-rich lipid bilayers designed to emulate native caveolar membranes. Site-specific structural constraints were obtained for the bound protein fragment, revealing a transition from an extended, β-stranded conformation to an α-helical structure near the central Tyr residue characteristic of CRAC motifs – consistent with certain pre-existing models of CRAC motif structure. Furthermore, complementary static and magic-angle-spinning (MAS) NMR measurements reveal the effects on lipid dynamics and indicate immersion of the polypeptide into the membrane hydrophobic core. These structural and motional data on both partners in this polypeptide-membrane interaction provide an unprecedented site-specific perspective on a cholesterol-binding CRAC motif.

Huntington's disease (HD) is one of ten neurodegenerative diseases caused by expanded CAG repeats. A characteristic feature of postmortem HD brains is the presence of intra-nuclear inclusions comprising N terminal mutant Huntingtin (htt) fragments. Based on these and other results, it was posited that protein aggregation might play a crucial role in mediating disease pathologies. Using exon-1 peptide models, we have been able to delineate a clear link between polyglutamine expansion and aggregation propensities as modulated by the first 17 residues adjacent to polyglutamine in the N terminus (httNT). Here we investigate the effect of httNT amino acid modifications - in particular mutations designed to block or mimic putative post-translational modification (PMTs) - on the aggregation of these exon-1 peptides. A particularly striking result was that exon-1 peptides in which both httNT serine residues are mutated to the phospho-Ser mimic aspartate aggregate more slowly and form irregular/immature aggregates, compared to peptides with WT httNT sequence. These results nicely correlate with results in a tg mouse model of HD, in which the Ser->Asp double mutant produces no aggregates and does not develop HD symptoms (X. W. Yang, personal communication). Analysis of single Ser to Asp mutants suggests that these mutations act in concert to produce these effects. Over the PMT mutations studied, we observed a correlation between net hydrophobicity and aggregation propensity. This observation was further corroborated in two multiple mutants containing mutations not associated with PMTs that are designed to either increase or suppress net hydrophobicity. We believe our data to date support our hypothesis that one to a few mutations or PMTs in the N terminal segment can have significant effects on the development of HD pathology, possibly mediated largely by biophysical effects.

The N-terminal 17 amino acid sequence in huntingtin (httNT) plays a crucial role in the aggregation of htt N-terminal fragment peptides (htt NTFs). In the current mechanistic model, httNT segments pack into α-helical bundles to form oligomers that create high local concentrations of appended polyglutamine (polyQ) segments, favoring nucleation of polyQ amyloid. Consistent with this model, we found previously that isolated httNT peptide added in trans inhibits the aggregation of htt NTF peptides, presumably by co-assembling with htt NTFs into mixed oligomers with decreased local polyQ concentrations. In a further test of this model, we found that the aggregation inhibitory activities of a set of ten scrambled httNT sequences correlate with their α-helical potential. We have now selected three of these scrambled sequences - (a) one that inhibits htt NTF aggregation (schttNTASSQ), (b) one that does not inhibit (schttNTFAKF), and (c) one that readily forms amyloid fibrils itself (unlike httNT or the other sequences) but does not inhibit (schttNTSAFM) - and used them in place of httNT to make synthetic analogs of htt NTF by adding a Q37P10K2 sequence. Consistent with expectations, we found that the peptide with the inhibitory, high α-helical potential, leader sequence (schttNTASSQ) exhibits an aggregation profile similar to that of the httNT-containing control peptide. In contrast, despite their identical amino acid compositions, htt NTF analogs containing the other two scrambled sequences (low aggregation inhibition, low α-helix) exhibit aggregation behavior more typical of simple polyQ sequences, hence deriving no kinetic benefit from their N-terminal sequences. Our results support a strong role for α-helix formation within httNT in greatly enhancing the kinetics of formation of the polyQ core of htt NTF amyloid.