Jean Baum

Folding of peptide models of collagen and misfolding in disease

The misfolding of the triple helix has been shown to play a critical role in collagen diseases. Normal and mutated collagen triple helices can be modeled by short, synthetic peptides of varying design. NMR spectroscopy and circular dichroism studies on the assembly of these peptide models have recently been used to isolate specific steps in the folding pathway and have provided information on the alterations resulting from mutations.

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

alpha-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinsons disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that alpha-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize alpha-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid beta component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.

Solution structure of a de novo protein from a designed combinatorial library

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries of de novo sequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designed a priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is “naïve” in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 is not a molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well folded de novo proteins.

Characterization of conformational and dynamic properties of natively unfolded human and mouse α-synuclein ensembles by NMR: implication for aggregation

Conversion of human alpha-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinsons disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinsons disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by (15)N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-A beta amyloid component (NAC) region that is both more exposed and has a higher propensity to beta structure may accelerate the rate of fibril formation of aS.

N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer.

The conformational properties of soluble α‐synuclein, the primary protein found in patients with Parkinsons disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N‐terminal acetylated α‐synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N‐terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue‐specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N‐terminal residues, as well as smaller long‐range changes in residues 28–31, 43–46, and 50–66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N‐terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α‐synuclein, but that their growth rates are slower. Our results highlight that N‐terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α‐synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.

Detection of Transient Interchain Interactions in the Intrinsically Disordered Protein α-Synuclein by NMR Paramagnetic Relaxation Enhancement

NMR paramagnetic relaxation enhancement experiments were applied to the intrinsically disordered protein alpha-synuclein, the primary protein in Parkinsons disease, to directly characterize transient intermolecular complexes at neutral and low pH. At neutral pH, we observed weak N- to C-terminal interchain contacts driven by electrostatic interactions, while at low pH, the C- to C-terminal interchain interactions are significantly stronger and driven by hydrophobic contacts. Characterization of these first interchain interactions will provide fundamental insight into the mechanism of amyloid formation.

Structure and dynamics of de novo proteins from a designed superfamily of 4-helix bundles

Libraries of de novo proteins provide an opportunity to explore the structural and functional potential of biological molecules that have not been biased by billions of years of evolutionary selection. Given the enormity of sequence space, a rational approach to library design is likely to yield a higher fraction of folded and functional proteins than a stochastic sampling of random sequences. We previously investigated the potential of library design by binary patterning of hydrophobic and hydrophilic amino acids. The structure of the most stable protein from a binary patterned library of de novo 4‐helix bundles was solved previously and shown to be consistent with the design. One structure, however, cannot fully assess the potential of the design strategy, nor can it account for differences in the stabilities of individual proteins. To more fully probe the quality of the library, we now report the NMR structure of a second protein, S‐836. Protein S‐836 proved to be a 4‐helix bundle, consistent with design. The similarity between the two solved structures reinforces previous evidence that binary patterning can encode stable, 4‐helix bundles. Despite their global similarities, the two proteins have cores that are packed at different degrees of tightness. The relationship between packing and dynamics was probed using the Modelfree approach, which showed that regions containing a high frequency of chemical exchange coincide with less well‐packed side chains. These studies show (1) that binary patterning can drive folding into a particular topology without the explicit design of residue‐by‐residue packing, and (2) that within a superfamily of binary patterned proteins, the structures and dynamics of individual proteins are modulated by the identity and packing of residues in the hydrophobic core.

Sequence Dependence of Renucleation after a Gly Mutation in Model Collagen Peptides

Missense mutations in the collagen triple helix that replace one Gly residue in the (Gly-X-Y)n repeating pattern by a larger amino acid have been shown to delay triple helix folding. One hypothesis is that such mutations interfere with the C- to N-terminal directional propagation and that the identity of the residues immediately N-terminal to the mutation site may determine the delay time and the degree of clinical severity. Model peptides are designed to clarify the role of tripeptide sequences N-terminal to the mutation site, with respect to length, stability, and nucleation propensity, to complete triple helix folding. Two sets of peptides with different N-terminal sequences, one with the natural sequence α1(I) 886-900, which is just adjacent to the Gly901 mutation, and one with a GPO(GAO)3 sequence, which occurs at α1(I) 865-879, are studied by CD and NMR. Placement of the five tripeptides of the natural α1(I) collagen sequence N-terminal to the Gly to Ala mutation site results in a peptide that is folded only C-terminal to the mutation site. In contrast, the presence of the Hyp-rich sequence GPO(GAO)3 N-terminal to the mutation allows complete refolding in the presence of the mutation. The completely folded peptide contains an ordered central region with unusual hydrogen bonding while maintaining standard triple helix structure at the N- and C-terminal ends. These peptide results suggest that the location and sequences of downstream regions favorable for renucleation could be the key factor in the completion of a triple helix N-terminal to a mutation.

A Revised Picture of the Cu(II)−α-Synuclein Complex: The Role of N‑Terminal Acetylation

α-Synuclein (αS) is an amyloidogenic intrinsically disordered protein implicated in Parkinsons disease, for which copper-mediated pathways of neurodegeneration have been suggested. We have employed nuclear magnetic resonance, circular dichroism, electrospray ionization mass spectrometry, and thioflavin T fluorescence to characterize interactions of Cu(2+) with the physiological acetylated form (Ac-αS). Significantly, N-terminal acetylation abolishes Cu(2+) binding at the high-affinity M1-D2 site present in the nonacetylated protein and maintains Cu(2+) interactions around H50/D121. Fibrillation enhancement observed at an equimolar Cu(2+) stoichiometry with the nonacetylated model does not occur with Ac-αS. These findings open new avenues of investigation into Cu(2+)-mediated neurodegenerative pathology suggested in vivo.

Conformational Features of a Natural Break in the Type IV Collagen Gly-X-Y Repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the α5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm = 29 °C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 °C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.