A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo.

/pdf/diffusion-crowding-protein-stability-in-a-dynamic-molecular-563ej6o48w.pdf

Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm.

The intracellular environment represents an extremely crowded milieu, with a limited amount of free water and an almost complete lack of unoccupied space. Obviously, slightly salted aqueous solutions containing low concentrations of a biomolecule of interest are too simplistic to mimic the “real life” situation, where the biomolecule of interest scrambles and wades through the tightly packed crowd. In laboratory practice, such macromolecular crowding is typically mimicked by concentrated solutions of various polymers that serve as model “crowding agents”. Studies under these conditions revealed that macromolecular crowding might affect protein structure, folding, shape, conformational stability, binding of small molecules, enzymatic activity, protein-protein interactions, protein-nucleic acid interactions, and pathological aggregation. The goal of this review is to systematically analyze currently available experimental data on the variety of effects of macromolecular crowding on a protein molecule. The review covers more than 320 papers and therefore represents one of the most comprehensive compendia of the current knowledge in this exciting area.

https://www.mdpi.com/1422-0067/15/12/23090/pdf

What Macromolecular Crowding Can Do to a Protein

There is extensive, yet fragmented, evidence of gender differences in academia suggesting that women are underrepresented in most scientific disciplines and publish fewer articles throughout a career, and their work acquires fewer citations. Here, we offer a comprehensive picture of longitudinal gender differences in performance through a bibliometric analysis of academic publishing careers by reconstructing the complete publication history of over 1.5 million gender-identified authors whose publishing career ended between 1955 and 2010, covering 83 countries and 13 disciplines. We find that, paradoxically, the increase of participation of women in science over the past 60 years was accompanied by an increase of gender differences in both productivity and impact. Most surprisingly, though, we uncover two gender invariants, finding that men and women publish at a comparable annual rate and have equivalent career-wise impact for the same size body of work. Finally, we demonstrate that differences in publishing career lengths and dropout rates explain a large portion of the reported career-wise differences in productivity and impact, although productivity differences still remain. This comprehensive picture of gender inequality in academia can help rephrase the conversation around the sustainability of women’s careers in academia, with important consequences for institutions and policy makers.

https://www.pnas.org/content/pnas/117/9/4609.full.pdf

Historical comparison of gender inequality in scientific careers across countries and disciplines.

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 1)?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 ...



Here, we attempt to answer these questions by reviewing the physical and biological properties of intracellular environments in relation to structural and functional parameters of disordered proteins. Specifically, we discuss how IDPs may experience in vivo environments differently to ordered proteins. To this end, we provide a description of the compositional and physical parameters of the cellular milieu and their effects on ordered and disordered proteins (section 2). We evaluate how biological processes may act differently on ordered and disordered proteins (section 3) and discuss how combined physical and biological contributions modulate the intracellular aggregation behavior of IDPs (section 4). Finally, we review theoretical and experimental approaches to study the structural and functional properties of disordered proteins in cells (section 5).

Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs).

Macromolecular Crowding In Vitro, In Vivo, and In Between

Thirty percent of a cell’s volume is filled with macromolecules, and protein chemistry in a crowded environment is predicted to differ from that in dilute solution. We quantified the effect of crow...

Protein crowding tunes protein stability.

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 1H 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 ...

/pdf/volume-exclusion-and-soft-interaction-effects-on-protein-1o4q8rdzlt.pdf

Volume exclusion and soft interaction effects on protein stability under crowded conditions.

Women have achieved parity with men among biomedical science degree holders but remain underrepresented in academic positions. The National Institutes of Health (NIH)—the world’s largest public funder of biomedical research—receives less than one-third of its new grant applications from women. Correspondingly, women compose less than one-third of NIH research grantees, even though they are as successful as men in obtaining first-time grants. Our study examined women’s and men’s NIH funding trajectories over time ( n = 34,770), exploring whether women remain funded at the same rate as men after receiving their first major research grants. A survival analysis demonstrated a slightly lower funding longevity for women. We next examined gender differences in application, review, and funding outcomes. Women individually held fewer grants, submitted fewer applications, and were less successful in renewing grants—factors that could lead to gender differences in funding longevity. Finally, two adjusted survival models that account for initial investigator characteristics or subsequent application behavior showed no gender differences, suggesting that the small observed longevity differences are affected by both sets of factors. Overall, given men’s and women’s generally comparable funding longevities, the data contradict the common assumption that women experience accelerated attrition compared with men across all career stages. Women’s likelihood of sustaining NIH funding may be better than commonly perceived. This suggests a need to explore women’s underrepresentation among initial NIH grantees, as well as their lower rates of new and renewal application submissions.

https://www.pnas.org/content/pnas/115/31/7943.full.pdf

NIH funding longevity by gender

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.

/pdf/protein-nuclear-magnetic-resonance-under-physiological-vlhq0dv247.pdf

Protein nuclear magnetic resonance under physiological conditions.

The biophysical properties of proteins in the crowded intracellular environment are expected to differ from those for proteins in dilute solution. Crowding can be studied in vitro through addition of polymers at high concentrations. NMR-detected amide 1H exchange is the only technique that provides equilibrium stability data for proteins on a per-residue basis under crowded conditions. We describe the theory behind amide 1H exchange and provide a detailed description of the experiments used to quantify globular protein stability at the residue level under crowded conditions. We also discuss the detection of weak interactions between the test protein and the crowding molecules.

Andrew C. Miklos

Papers

Protein crowding tunes protein stability.

Volume exclusion and soft interaction effects on protein stability under crowded conditions.

NIH funding longevity by gender

Protein nuclear magnetic resonance under physiological conditions.

Using NMR-detected backbone amide 1H exchange to assess macromolecular crowding effects on globular-protein stability.