Interaction of α-Synuclein and a Cell Penetrating Fusion Peptide with Higher Eukaryotic Cell Membranes Assessed by 19 F NMR

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a 19F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of 19F resonances of 3-fluoro-L-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts

Residue level quantification of protein stability in living cells

Proteins function in a sea of macromolecules within cells, but are traditionally studied under ideal conditions in vitro. The more details we amass from experiments performed in cells, the closer we will get to understanding fundamental aspects of protein chemistry in the cellular environment. In addition to furthering our essential knowledge of biochemistry, advancements in the field of macromolecular crowding will drive efforts to stabilize protein-based therapeutics. Here, we show that protein stability can be measured at the residue level in living cells without adding destabilizing cosolutes or heat

Impact of reconstituted cytosol on protein stability

The cell cytoplasm contains a complex array of macromolecules at concentrations exceeding 300 g/L. The natural, most relevant state of a biological macromolecule is thus a “crowded” one. Moving quantitative protein chemistry from dilute solution to the inside of living cells represents a major frontier that will affect not only our fundamental biological knowledge, but also efforts to produce and stabilize protein-based pharmaceuticals. We show that the bacterial cytosol actually destabilizes our test protein, contradicting most theoretical predictions, but in agreement with a novel Escherichia coli model

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

Abstractα-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and 19F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The 19F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes

FlgM gains structure in living cells

Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (≥400 g/liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions

Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions

Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams per liter of macromolecules often interfere with observing the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyze the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues where stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions need to be seriously considered when applying both theory and experiment to studies of macromolecular crowding

α-Synuclein Conformation Affects Its Tyrosine-Dependent Oxidative Aggregation †

Oxidative stress and aggregation of the protein α-synuclein are thought to be key factors in Parkinson’s disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including α-synuclein. Here, we examine the role of each of α-synuclein’s four tyrosine residues and how the protein’s conformation affects covalent oxidative aggregation. When α-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when α-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson’s disease

α-Synuclein Conformation Affects Its Tyrosine-Dependent Oxidative Aggregation †

Oxidative stress and aggregation of the protein α-synuclein are thought to be key factors in Parkinson’s disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including α-synuclein. Here, we examine the role of each of α-synuclein’s four tyrosine residues and how the protein’s conformation affects covalent oxidative aggregation. When α-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when α-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson’s disease

A bioreactor for in-cell protein NMR

The inside of the cell is a complex environment that is difficult to simulate when studying proteins and other molecules in vitro. We have developed a device and system that provides a controlled environment for Nuclear Magnetic Resonance (NMR) experiments involving living cells. Our device comprises two main parts, an NMR detection region and a circulation system. The flow of medium from the bottom of the device pushes alginate encapsulated cells into the circulation chamber. In the chamber, the exchange of oxygen and nutrients occurs between the media and the encapsulated cells. When the media flow is stopped, the encapsulated cells fall back into the NMR detection region, and spectra can be acquired. We have utilized the bioreactor to study the expression of the natively disordered protein α-synuclein, inside Escherichia coli cells

Protein Nuclear Magnetic Resonance under Physiological Conditions †

Almost everything we know about protein biophysics comes from studies on purified proteins in dilute solution. Most proteins, however, operate inside cells where the concentration of macromolecules can be >300 mg per mL. Although reductionism-based approaches have served protein science well for over a century, biochemists now have the tools to study proteins under these more physiologically-relevant conditions. We review a part of this burgeoning post-reductionist landscape by focusing on high-resolution protein NMR spectroscopy, the only method that provides atomic-level information over an entire protein under the crowded conditions found in cells