Gregory B. Young

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.

Solvent‐induced collapse of α‐synuclein and acid‐denatured cytochrome c

The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, α‐synuclein and acid‐denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of α‐synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that α‐synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid‐denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that α‐synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.

Unexpected Effects of Macromolecular Crowding on Protein Stability

Most theories about macromolecular crowding focus on two ideas: the macromolecular nature of the crowder and entropy. For proteins, the volume excluded by the crowder favors compact native states over expanded denatured states, enhancing protein stability by decreasing the entropy of unfolding. We tested these ideas with the widely used crowding agent Ficoll-70 and its monomer, sucrose. Contrary to expectations, Ficoll and sucrose have approximately the same stabilizing effect on chymotrypsin inhibitor 2. Furthermore, the stabilization is driven by enthalpy, not entropy. These results point to the need for carefully controlled studies and more sophisticated theories for understanding crowding effects.

Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy

In-cell NMR provides a valuable means to assess how macromolecules, with concentrations up to 300 g/L in the cytoplasm, affect the structure and dynamics of proteins at atomic resolution. Here an intrinsically disordered protein, alpha-synuclein (alphaSN), and a globular protein, chymotrypsin inhibitor 2 (CI2) were examined by using in-cell NMR. High-resolution in-cell spectra of alphaSN can be obtained, but CI2 leaks from the cell and the remaining intracellular CI2 is not detectable. Even after stabilizing the cells from leakage by using alginate encapsulation, no CI2 signal is detected. From in vitro studies we conclude that this difference in detectability is the result of the differential dynamical response of disordered and ordered proteins to the changes of motion caused by the increased viscosity in cells.

Residue-Level Interrogation of Macromolecular Crowding Effects on Protein Stability

Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.

Temperature‐induced reversible conformational change in the first 100 residues of α‐synuclein

Natively disordered proteins are a growing class of anomalies to the structure–function paradigm. The natively disordered protein α‐synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinsons disease. We noticed a dramatic difference in dilute solution 1H‐15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild‐type α‐synuclein and two disease‐related mutants (A30P and A53T), with spectra collected at 35°C showing fewer cross‐peaks than spectra acquired at 10°C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing α‐synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H‐15N HSQC spectra obtained at 35°C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of α‐synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.

Expression of 15N-labeled eukaryotic cytochrome c in Escherichia coli.

Abstract We present a simple and inexpensive method for producing 15N-labeled Saccharomyces cerevisiaeiso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra.

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 alpha-synuclein, inside Escherichia coli cells.

Amide proton exchange of a dynamic loop in cell extracts

Intrinsic rates of exchange are essential parameters for obtaining protein stabilities from amide 1H exchange data. To understand the influence of the intracellular environment on stability, one must know the effect of the cytoplasm on these rates. We probed exchange rates in buffer and in Escherichia coli lysates for the dynamic loop in the small globular protein chymotrypsin inhibitor 2 using a modified form of the nuclear magnetic resonance experiment, SOLEXSY. No significant changes were observed, even in 100 g dry weight L−1 lysate. Our results suggest that intrinsic rates from studies conducted in buffers are applicable to studies conducted under cellular conditions.

Protein shape modulates crowding effects

Significance Macromolecular crowding influences protein−protein interactions via hard-core repulsions and chemical interactions. Scaled particle theory predicts that the effect of hard-core repulsions depends on the shape of the protein complex, and simple ideas about chemical interactions predict a dominant role for the chemical qualities of the protein surface. The theory predicts that a collapsed, ellipsoidal, dimer will be stabilized by hard-core repulsions, whereas a less collapsed, side-by-side, dimer will not. We applied scaled particle theory to two dimers with nearly identical surfaces but different shapes. Our results support the predictions; crowders that interact primarily through hard-core repulsions stabilize the ellipsoidal domain-swapped dimer more than the side-by-side dimer, whereas crowders that interact via chemical interactions have the same effect on both dimers. Protein−protein interactions are usually studied in dilute buffered solutions with macromolecule concentrations of <10 g/L. In cells, however, the macromolecule concentration can exceed 300 g/L, resulting in nonspecific interactions between macromolecules. These interactions can be divided into hard-core steric repulsions and “soft” chemical interactions. Here, we test a hypothesis from scaled particle theory; the influence of hard-core repulsions on a protein dimer depends on its shape. We tested the idea using a side-by-side dumbbell-shaped dimer and a domain-swapped ellipsoidal dimer. Both dimers are variants of the B1 domain of protein G and differ by only three residues. The results from the relatively inert synthetic polymer crowding molecules, Ficoll and PEG, support the hypothesis, indicating that the domain-swapped dimer is stabilized by hard-core repulsions while the side-by-side dimer shows little to no stabilization. We also show that protein cosolutes, which interact primarily through nonspecific chemical interactions, have the same small effect on both dimers. Our results suggest that the shape of the protein dimer determines the influence of hard-core repulsions, providing cells with a mechanism for regulating protein−protein interactions.