Conggang Li

Effects of Proteins on Protein Diffusion

Despite increased attention, little is known about how the crowded intracellular environment affects basic phenomena like protein diffusion. Here, we use NMR to quantify the rotational and translational diffusion of a 7.4-kDa test protein, chymotrypsin inhibitor 2 (CI2), in solutions of glycerol, synthetic polymers, proteins, and cell lysates. As expected, translational diffusion and rotational diffusion decrease with increasing viscosity. In glycerol, for example, the decrease follows the Stokes-Einstein and Stokes-Einstein-Debye laws. Synthetic polymers cause negative deviation from the Stokes laws and affect translation more than rotation. Surprisingly, however, protein crowders have the opposite effect, causing positive deviation and reducing rotational diffusion more than translational diffusion. Indeed, bulk proteins severely attenuate the rotational diffusion of CI2 in crowded protein solutions. Similarly, CI2 diffusion in cell lysates is comparable to its diffusion in crowded protein solutions, supporting the biological relevance of the results. The rotational attenuation is independent of the size and total charge of the crowding protein, suggesting that the effect is general. The difference between the behavior of synthetic polymers and protein crowders suggests that synthetic polymers may not be suitable mimics of the intracellular environment. NMR relaxation data reveal that the source of the difference between synthetic polymers and proteins is the presence of weak interactions between the proteins and CI2. In summary, weak but nonspecific, noncovalent chemical interactions between proteins appear to fundamentally impact protein diffusion in cells.

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.1−4 The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,5−7 and that it is often synonymous with function,8−11 disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways12 and functions as a prominent player in many regulatory processes.13−15 Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.16 They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.17,18 Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles3,19,20 and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.1,21 The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions22 and high-resolution insights into the architectures of amyloid fibrils were obtained.23,24 On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.15,25,26 One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure ​(Figure11)?27−29 Figure 1 Intracellular complexity. (A) Left: Cryo-electron tomography slice of a mammalian cell. Middle: Close-up view of cellular structures colored according to their identities: Right: Three-dimensional surface representation of the same region. Yellow, endoplasmic ...

Protein 19F NMR in Escherichia coli

Although overexpression and (15)N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, (15)N enrichment alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using (19)F-labeled proteins. Resonances from small (approximately 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the (19)F resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using (19)F NMR.

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.

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 of macromolecules per liter often interfere with the observation of 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 (1)H exchange. We analyzed 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 for which 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 needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.

Azobenzene‐Based Colorimetric Chemosensors for Rapid Naked‐Eye Detection of Mercury(II)

Two new highly selective colorimetric chemosensors for Hg(2+), based on azobenzene and highly selective Hg(2+)-promoted deprotection of a dithioacetal have been designed and synthesized. In the presence of as little as 20 μM Hg(2+), the sensors change their color from light yellow to deep red, which can easily be observed by the naked eye. The underlying signaling mechanism is intramolecular charge transfer (ICT). The sensors have good selectivity for Hg(2+) with respect to several common alkali, alkaline earth, and transition metal ions. Furthermore, they can be used for in-the-field measurements by virtue of a dipstick approach without any additional equipment.

Soft interactions and crowding

The intracellular milieu is complex, heterogeneous and crowded—an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.

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/mL. Although reductionism-based approaches have served protein science well for more than a century, biochemists now have the tools to study proteins under these more physiologically relevant conditions. We review a part of this burgeoning postreductionist landscape by focusing on high-resolution protein nuclear magnetic resonance (NMR) spectroscopy, the only method that provides atomic-level information over an entire protein under the crowded conditions found 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.

In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State Magic-Angle-Spinning NMR

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimers disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure.