New imaging techniques down to the picometer scale are permitting the detection of previously unknown alliances of cellular software with electrostatics and mechanics. Such knowledge was unattainable until biophysicists gained the ability to measure phenomena at the atomic level. What they are finding multiplies the information content embedded in the molecules of life.

Early depictions of molecules in the nucleus showed them drifting around aimlessly. How could molecules do otherwise without membranes to hold them together? Organelles are defined by their lipid membranes. The simplified picture of molecules in lipid cages, like animals in a zoo, raised questions about how enzymes locate their substrates in regions that, at their scale, would be distant. Last December, we reported findings at Caltech that revealed smaller levels of organization at play: nuclear speckles, transcriptional condensates and other “membraneless organelles” coordinated by non-coding RNAs. These erstwhile “junk” parts of the genome turned out to play key roles in architecting the “office layout” of the cellular factory. Some ncRNAs actually recruit the partners needing to associate like managers calling a meeting.

The Electric Cell

New findings reported in PNAS by Toyama et al. are uncovering a role for electrostatics in enzymatic activity. Simultaneously, the discovery may offer insight into the function of so-called “disordered proteins” that never fold into stable structures, and other proteins containing disordered regions that would seem to flail about like loose cables. But there is order in the disorder! How big is this discovery?

Electrostatic interactions play important roles in regulating a plethora of different biochemical processes and in providing stability to biomolecules and their complexes

What the team from the University of Toronto found, discussed below, was only made possible by “solution NMR spectroscopy.” This technique allows them, for the first time, to measure the near-surface electrostatic potentials of individual atoms in proteins and follow changes in those potentials during an enzyme’s action.

Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates. [Emphasis added.]

CAPRIN1 (cell cycle associated protein 1) is an RNA-binding protein “localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation.” It may act as a negative regulator of translation, confining mRNAs in condensates at times to prevent overproduction of proteins. “CAPRIN1 is found in membraneless organelles, such as stress granules, P bodies, and messenger RNA (mRNA) transport granules, where, in concert with a variety of other RNA-binding proteins, it plays an important role in regulating RNA processing,” the paper explains. In humans, this enzyme appears associated with long-term memory through the regulation of dendritic spine density. If so, our memories are not just dependent on chemistry, but on electrostatics, too.

CAPRIN1 contains IDP tails at both ends which, it turns out, are the key to condensate formation. The Toronto team found, importantly, that ATP plays a dynamic role in the electrostatic changes of CAPRIN1, especially in its IDP regions. In brief, here is what happens (see Figure 5 in the paper). Specific amino acid residues in the IDP regions confer on them a net positive charge. This makes the tails repel each other, resisting condensate formation (and preventing self-association of the tails). When ATP attaches to the IDP regions, however, the net charge is reduced, permitting intermolecular interactions. As more ATP is added, the collection becomes neutral, and a condensate forms. Additional ATP inverts the electrical potential, making it negative. Electrostatic repulsion ensues again, causing breakup of the condensate, separating the contents and freeing them up for the next round. 

This implies that condensate formation has an electrical aspect to it. Since it relies on the sequence and position of specific amino acid residues, one might even call it an electric code.

Our interest in these experiments lies in applications to intrinsically disordered proteins (IDPs) and to intrinsically disordered regions (IDRs) of proteins, collectively referred to as IDPs in what follows. It is estimated that ∼30% of residues within human proteins encode regions of disorder, comprising at least 30 amino acids, with many of these proteins playing critical roles in cellular function, including modulating the formation of membraneless biomolecular condensates that organize proteins and/or nucleic acids, along with a variety of small molecules to regulate biochemical processes in the cell. At least 75% of IDPs contain both positively and negatively charged residues, with charge–charge interactions important in defining their physical and chemical properties and, in some cases, their propensities to phase separate.

The information in the sequence of amino acids, and of the codons in the genes that encode them, appears to play critical roles in condensate formation and, simultaneously, in enzymatic behavior. Some amino acids they dub “stickers” promote phase separation. The specific electrostatic attractions and repulsions that give rise to the enzyme’s function during condensate formation and dissolution is dependent on the positions of these stickers.

This remarkable revelation begins to give insight into the participation of cell coding with electrophysics. Get a charge out of that!

CAPRIN1 coexists with negatively charged RNA molecules in cells and, along with FMRP and other proteins, is implicated in the regulation of RNA processing and translational activity. Thus, electrostatics play a central role in modulating the biological functions of this protein, and measurement of electrostatic potentials at each site along its backbone, as reported here, provides an opportunity to understand in more detail the important role of charge in this system.

The paper only investigated one enzyme, so caution is advised before generalizing. The authors feel, though, that this electrical code model will help explain many other processes that require molecules to come together, perform their work, and then separate. It’s the new Electric Cell.

Future applications of these methods will pave the way for mapping the role of electrostatics in phase separation in a more general sense, including the effects of sequence, charge patterning, posttranslational modifications, and the presence of nucleic acids.

Coded Mechanics, Too

Another case of physics in cellular processing was uncovered by a team from the University of Washington who also published their work in PNAS. And once again, it was new creative imaging at the atomic scale that made the discovery possible.

This team worked on a helicase enzyme named PcrA, which unwinds DNA for transcription. This enzyme works so fast (1000 bases per second!) it’s been like trying to describe the blur of a racecar speeding down a track. Using a new technique called “single-molecule picometer-resolution nanopore tweezers” (SPRNT), they were able to slow down the action and watch the racecar move with its “inchworm mechanism” one base at a time. This blends chemistry with another branch of physics, mechanics: “mechanochemistry.”

We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion.…Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

The authors are not clear why this is. What is evolution up to? They figure that there must be a reason.

Unlike protein filaments (e.g., actin), DNA is not a homogeneous track; sequence-dependent behavior may be the norm rather than the exception. Strong sequence-dependent enzyme kinetics such as those observed in our data likely affect PcrA’s role in vivo and could thereby exert selective pressure on both DNA and protein evolution. Therefore, sequence-dependent behavior should be carefully considered in future studies of any enzyme that walks along DNA or RNA, since the sequence-dependent kinetics may reveal essential features of an enzyme’s function. Such effects are almost certainly used by life to achieve various ends, and SPRNT is well suited to discovering how and why such sequence dependence occurs and opens the possibility of uncovering enzyme functions that were hereto unknown.

Why are they giving the credit to blind evolution? If life uses “sequence-dependent kinetics…to achieve various ends,” that sounds like intelligent design, not evolution. Design advocates are accustomed to forgiving logical malapropisms like this. They look past the magical thinking and see the operation of a designing mind with foresight and purpose, intimately familiar with the laws of physics, able to write code to utilize those laws in precision operations. Now, it becomes clear that the precision goes deeper than previously known.