The Dynamic Dance of Protein Charge and its Biological Importance

The location and magnitude of a protein’s charge is really important for its biological functions. However, protein charge is not always fixed, but rather, they can be controlled by many mechanisms that occur over a wide range of timescales. And this dynamic control of protein charge turns out to also be really important in protein function.

When talking about protein charge, we’re starting to get deeper into the world of proteins, and it may have you wondering why it matters or what is the importance of protein charge.

A protein’s overall charge, and where these charges are distributed throughout the protein, is crucial for interactions with other biomolecules and the protein’s function. But, a protein’s charge is not set. Rather, different biological factors tune protein charge across broad periods.

In this article we will discuss how a range of different mechanisms – including posttranslational modifications, proteolytic cleavage, environmental pH, RNA-spicing variants, and evolutionary mutations – are used to change a protein’s charge. As you’ll see these simple changes in charge build in, or erase, molecular interactions that have key roles in maintaining homeostasis and causing disease.

In this article:

Biological Importance of Protein Charge

Ways to Change a Protein’s Charge

Posttranslational Modifications

Change in Environmental pH

Splice Variants

Evolutionary Changes to Protein Sequences

References

Biological Importance of Protein Charge

Proteins interact with other proteins, nucleic acids, lipids, and small molecules to carry out their function. Charged residues are enriched on the surface of proteins and make key interactions with other biomolecules (Grassmann et al., 2023).

For example, positive residues are enriched at protein surfaces that bind to negatively charged DNA, RNA, and lipid molecules (Mishra and Levy, 2015; Pérez-Cano & Fernández-Recio, 2010; Thakur et al, 2023; Weber and Steitz, 1984) (Figure 1).

Additionally, many proteins are enzymes that catalyze chemical reactions, and charged residues are very important for these functions. Charged residues are also key residues at the active sites of enzymes that directly contact reactants and cofactors and catalyze chemical reactions (Sheinerman et al, 2000).

So protein charge is really important for molecular interactions and enzymatic function. That might make you think that cells should be really careful to make sure that a protein’s charge never changes. However, for many proteins it’s actually quite the opposite. Modulating a protein’s charge, through the mechanisms described below, allows the cell to rapidly toggle particular functions and signaling pathways on and off, and back on again!

Figure 1. Protein charge is important in interactions with DNA (left), RNA (middle), and lipids (right). DNA, RNA and lipid are colored gray. Protein is colored according to charge in left and middle images – blue is positive and red is negative charge. In right image an arginine residue (blue spheres) is important for the protein’s (green) interaction with negatively charged lipid head groups (red). Structures from PDB files 4IRI, 3SNP, and 1LN1.

Ways to Change a Protein’s Charge

Static images often give the impression of biology being no more alive than the painting of Mona Lisa. Yet, in reality, molecules, cells, tissues, and organisms are vibrantly dynamic systems (Figure 2).

For example, protein charge is not set in stone, but rather is a parameter that can be tuned in response to the surrounding environment. Protein charge is rapidly modified on the order of seconds to minutes by:

• Changes in environmental pH
• Posttranslational modifications
• Proteolytic cleavage
• mRNA splicing variants

More gradually over the course of minutes to hours a protein’s charge is changed by:

• And on really slow evolutionary timescales of millions of years, protein charge is altered by:

• genetic mutations

Figure 2. Timescales for various changes in protein charge.

Posttranslational Modifications

Recall from our article on calculating protein charge that tyrosine phosphorylation and lysine acetylation are two posttranslational modifications that change the charge of those amino acid’s side chains (Figure 3).

Figure 3. Phosphorylation of tyrosine residues and acetylation of lysine residues are dynamic and reversible modifications that change the charge on their side chains.

One way that these charge-changing modifications impact protein function is by forming new binding sites for other proteins.

For example, SH2 (Src homology 2) protein domains specifically bind to phosphorylated tyrosine residues (Figure 4). Phosphorylation creates a new binding site that is important for transmitting cellular signaling pathways. Since phosphorylation is fast and removable, phosphorylation essentially serves as a toggle to dynamically turn on and off cellular signaling pathways.

Figure 4. SH2 domains bind to phosphorylated tyrosine residues. The surface of an SH2 domain is colored according to its electrostatic charge with blue representing positive residues and red negative residues. A peptide (gray) with a negatively charged phosphorylated tyrosine (magenta) binds to a positively charged patch on SH2. I made this figure in PyMol using structures 1SPR and 1SPS from the Protein Data Bank (Wakman et al, 1993).

Similarly, there are other proteins that specifically bind to acetylated lysine residues (Figure 5).

Figure 5. Acetylated lysine residues bind to bromodomains. The bromodomain of the protein BAZ2B is shown in surface rendering and colored according to charge as in Figure 4. Acetylated lysine (orange spheres) binds to a hydrophobic pocket on the bromodomain. Acetylation masks the positive charge of lysine making this interaction more energetically favorable. I made this figure in PyMol using structure 4NR9 from the Protein Data Bank (Ferguson et al, 2013).

Acetylated lysine residues are important in a wide range of biological disciplines, including epigenetics. Epigenetics literally means “above genetics,” and refers to things that control which genes are being expressed at any given time in any given cell. Acetylation marks on histone proteins help recruit other proteins that turn on the transcription of nearby genes into messenger RNA.

Like tyrosine phosphorylation, lysine acetylation is also reversible. This makes histone acetylation a dynamic process that regulates nearby gene expression.

Gene expression is really important in cell identity, which is a fancy way of saying that a different set of genes are expressed to make a liver cell and a skin cell, for example, different.

Histone acetylation and downstream gene expression can be misregulated in diseases such as cancer. Indeed proteins that write, erase, and read histone acetylation marks are implicated in many diseases including several different types of cancer. Writers are enzymes that acetylate histone tails, erasers are enzymes that take the acetyl mark off histone tails, and readers are proteins that bind to acetylated histones to recruit other proteins to that site in the genome.

The good news is that since we know what is going wrong in these diseases, we can try to fix it! Several drugs that inhibit the above mentioned proteins are already used in the clinic and even more are undergoing clinical trials for many types of cancer (Jones et al, 2016; White et al, 2024)(Table 1).

Table 1. Cancer drugs modulating histone acetylation.

Protein type	Protein	Drug	Disease
Histone deacetylase	Class I HDACs	Romidepsin	T-cell lymphoma
Histone deacetylase	Pan-HDAC	Belinostat	Peripheral T-cell lymphoma
Histone deacetylase	Pan-HDAC	Vorinostat	Cutaneous T-cell lymphoma
Histone deacetylase	Pan-HDAC	Panobinostat	Multiple myeloma
Histone deacetylase	LSD1	Iadademstat	Acute myeloid leukemia
Histone acetyltransferase	KAT6A/B	PF-07248144	Metastatic breast, prostate, and lung cancer
Acetyl reader	CBP/P300	EP31670	Metastatic prostate cancer and NUT midline carcinoma

(Jones et al, 2016; White et al, 2024)

Proteolytic Cleavage

Do you like being jam packed in a busy crowd, like at the airport or on a rush hour subway for example? Yeah, me neither. Just like us, bacteria can sense how crowded their surroundings are through a process called quorum sensing.

One important protein for quorum sensing in the bacterium Pseudomonas aeruginosa is the protease leucine aminopeptidase, or LAP for short.

LAP is secreted from bacteria, and when bacteria get too crowded LAP gets activated and starts cleaving proteins which signals to bacteria to halt cell division because it is already too crowded. However, if LAP were always active then it would never allow the bacteria to grow even when it is not crowded. So how does LAP know when to turn on?

Well, LAP is translated in an inactive form. It has a positively charged C-terminal domain that interacts with its negatively charged catalytic site (Figure 6). This interaction prevents LAP from cutting up other proteins when bacteria are sparse (Sarnovsky et al, 2009).

Figure 6. When bacteria are sparse, LAP is in an inactive conformation where a positively charged inhibitory domain (magenta) interacts with the negatively charged catalytic domain (blue). When bacteria cells get crowded, the inhibitory domain is cut off of LAP, activating its activity which tells the cells to stop growing. See Sarnovsky et al, 2009 and the main text for more details.

When bacteria become overgrown, the positively charged C-terminal domain is cut off of LAP and now its own protease activity becomes supercharged. LAP cuts up several external bacterial proteins which tells these cells to stop growing.

This is a pretty nice example of how external cues can regulate a protein’s function. In this case since the catalytic domain is negatively charged and the inhibitory domain is positively charged, cutting off the positive domain will have a pretty big impact on the protein’s charge as well as its function.

Change in Environmental pH

When you think of biologically relevant pH, what number do you think of? Go ahead, I’ll play the Jeopardy theme song music while I wait …

Ok, pencils down. You probably came up with something like pH 7 to 7.4, right? This number has been drilled into our collective heads. And it isn’t wrong - the cytoplasm of many human cells is at pH ~ 7.1 to 7.2, which is why we always remember this range.

But all of the universe is not human cytoplasm, my friend!

In reality, biologically relevant pH spans a much wider range, from at least pH 4.5 to 8.5. Remember that pH is a log scale, so this 4-unit difference corresponds to a 10,000 (10⁴) fold difference in H⁺ concentration!

Microorganisms live in extreme acidic and basic conditions in environments such as ocean vents and alkaline lakes. pH can also differ between different parts of our bodies – the stomach is very acidic (pH 2) whereas the pancreas is slightly basic (pH 8). Even within individual cells, organelle pH can vary from the acidic lysosome (pH 4.5) to the basic mitochondria (pH 8).

Variety is the spice of life, and biology has a rich pH profile. Contrary to the bland pH 7 answer that is stuck in our head, biologically relevant pH actually varies from at least 4.5 to 8.5 depending on the organism, cellular, and subcellular location (Decker et al, 2021; Joyner et al, 2016; King et al, 2024, Munder et al, 2016; Rampelotto, 2013; White et al, 2017).

Remember that environmental pH regulates the charge on ionizable protein side chains. Therefore, when proteins or cells encounter different pH environments, protein charge will change.

pH-mediated changes in protein charge influence protein function by different mechanisms including:

• Modulating protein oligomerization state (Currie et al, 2023; Politi et al, 2009)
• Altering protein stability (Chiariello et al, 2023; White et al, 2018)
• Influencing interactions with other biomolecules (Yao et al, 2020)
• Changing enzymatic function (White et al, 2017)

Splice Variants

RNA splicing produces different forms of a protein that can have different charge states. What is RNA splicing, and how does it perform this magic?

When genes are transcribed, they are first turned into pre-mRNA. You may remember that pre-mRNA includes both exons and introns. Some exons are retained during processing of pre-mRNA into mRNA, and those exons are then translated into proteins. However, RNA splicing can change which exons are included and which ones are not (Figure 7). Introns, on the other hand, are spliced out of mRNAs and do not encode protein sequences.

The electrostatic properties of a protein can change substantially depending on which exons are included.

An interesting example of this are glutamate receptors. Glutamate receptors channel cations like sodium (Na⁺) and calcium (Ca²⁺) into and out of neurons. This is the language by which neurons communicate with one another, and how neuronal circuits are formed that allow us humans to move, learn, talk to other people – basically everything that we do!

Figure 7. Overview of RNA splicing. Pre-RNA contains both introns and exons. Some exons are included in the mRNA, whereas all introns and some exons are spliced out of the mRNA. The mRNA is then translated into protein. Therefore, you can get different, yet related, protein variants through RNA splicing.

So, if they channel cations, why are they called glutamate receptors? Glutamate binding to these receptors is what turns them on and off.

Typically, in the absence of glutamate, the receptor will be in the off state, blocking ions from passing through (Figure 8). Once glutamate binds, the channel will briefly turn on and allow ions to pass through. The channel will close while glutamate is still bound, and this is called the desensitized state because the first glutamate molecule needs to dissociate from the receptor before another glutamate can bind to the channel to activate it again.

Figure 8. Shows the channel activation cycle.

Here is a quick look at the process following figure 8. An ion channel (blue) will be in the off state, and ions (orange pentagons and hexagons) cannot pass through a membrane when its agonist, glutamate (magenta) in this case, is not bound to it (top). When glutamate binds, the channel briefly opens and passes ions through the membrane (bottom left). Glutamate will remain bound after the channel closes and the channel is desensitized as another glutamate cannot bind and activate the channel while the original glutamate is still bound (bottom right). Eventually glutamate will dissociate from the channel, and it will reset back to the off state (top).

One particular glutamate receptor, named GluK1, has a protein loop that can either be included in the final protein, or it can be spliced out. This loop is really intriguing from a charge standpoint because it is loaded with positive lysine and histidine residues (Figure 9).

Researchers found that this loop is really important in the desensitization state of GluK1. GluK1, with the positively charged loop, stays in the desensitized state longer compared to GluK1 without the loop (Dhingra et al, 2023). Therefore, the positive loop slows down the conversion rate between active and inactive channel states meaning this splice form will have lower overall ion channeling activity relative to the splice isoform without the loop (Figure 9).

Figure 9. A positively charged loop is included in one splice variant of the protein GluK1 (left). This variant will have slower activity than the splice variant without the loop (right) because it remains in the desensitized state (Figure 9) longer.

Obviously, we are skimming the surface here, but the key takeaway point is that simple changes in protein charge due to RNA splicing make a big difference in the molecular processes that underlie our brain’s function!

Evolutionary Changes to Protein Sequences

Protein charge also changes slowly over millions of years via genetic mutations that alter a protein’s primary sequence. To explain why these changes are important, we first need to cover a little background.

In multicellular organisms, such as humans, many proteins are part of what is known as protein families.

Think about your own family, there are probably a few ways in which you are similar to some of your family members – maybe it’s your height, your hair, or the way that you laugh. Yet, you’re not identical to any of your family members either. There are unique things about you that make you, you.

Similarly, protein families are classified by proteins that have similar structures and functions, yet they aren’t quite identical to one another either. Here, we’ll discuss two types of protein families:

• transcription factors that regulate gene expression
• kinases that phosphorylate other proteins

For example, proteins in a transcription factor family all have a similar domain that binds to DNA, and they will all regulate gene expression at the level of transcribing our genes into mRNA. Yet they all have slightly different ways in which they perform this function.

Distinct protein charge is one way proteins evolve unique biochemical and cellular functions. Let’s consider the transcription factor family again. Two different proteins in this family, on their own, bind to very similar DNA sequences using a conserved and positively charged DNA-binding surface (Figure 10). For simplicity let’s call these proteins blue and magenta.

Figure 10. Evolutionarily-related transcription factors magenta and blue bind to DNA using a conserved positively charged surface (left, top and bottom). Both transcription factors promote transcription at normal prostate genes as indicated by the green traffic lights. Magenta has a complementary negatively charged surface which allows the positively charged chopsticks transcription factor to also bind (top right) whereas the positively charged blue tumor suppressor prevents chopsticks from binding (bottom right). This difference in charge causes the difference in transcription of prostate cancer genes.

Yet, it is known that these two proteins have the exact opposite phenotype in prostate cancer. Magenta is an oncogene that promotes cancer formation and progression, and the blue is a tumor suppressor that prevents these processes (Albino et al, 2012; Cangemi et al, 2008; Tomlins et al, 2005; Tomlins et al, 2006). How in the world can two very similar proteins with nearly identical DNA-binding properties have the complete opposite effect on prostate cancer?

In part it is due to surfaces where those proteins have different electrostatic properties (Madison et al, 2018). In prostate cells magenta and blue bind to DNA sequences right next to a different type of transcription factor that looks like a pair of chopsticks (Hollenhorst et al, 2011). The chopstick transcription factors are very positively charged all over.

The magenta oncogene is neutral and negatively charged on its side that faces the chopsticks transcription factor, so they can both bind to DNA at the same time and increase the transcription of genes that are important for prostate cancer progression.

The blue tumor suppressor transcription factor, on the other hand, is also positively charged all over. You’ll remember that similar charges repel one another and so the tumor suppressor and chopsticks transcription factors cannot bind to DNA at the same time. The lower occupancy of these proteins reduces the transcription of prostate cancer related genes as compared to the red oncogene (Budka et al, 2018).

And so the punchline is that evolutionary changes over millions and millions of years create different charge properties allowing proteins with very similar structures and functions to have the exact opposite impact on cellular and organismal phenotypes. All it takes is a little charge - pretty cool, right.

There are protein families with all kinds of biological functions. One other example where changes in charged surfaces play an important role is protein kinases.

T cells are important immune cells, and the T cell receptor binds to foreign antigens. The binding of foreign particles is signaled through the T cell receptor and a series of downstream molecular interactions to trigger an effective immune response and eliminate the foreign pathogen. Kinases are key transducers of T cell signaling, and you’ll remember that kinases function by phosphorylating other proteins.

Phosphorylated tyrosine residues bind to SH2 domains, as we discussed above (Figure 4), and these interactions are critical for T cell signaling. One of the kinases in this pathway, ZAP-70, has a positively charged active site and so it prefers to phosphorylate tyrosine residues that are surrounded by negatively charged amino acids (Figure 11) (Shah et al, 2016). This preference encourages ZAP-70 to phosphorylate other kinases in the T cell signaling pathway instead of just phosphorylating itself.

Figure 11. Zap-70 has a positively charged active site giving it a preference for substrates with negative charges surrounding the tyrosine residue that will be phosphorylated.

Ok, we’re getting detailed here, so why does any of this matter? I’m so glad you asked!

If ZAP-70 phosphorylated itself just as efficiently as it does the other kinases in the pathway, then T cell signaling would always be active regardless of whether a foreign pathogen is present or not.

Autoactivation of T cell signaling in the absence of foreign invaders is one of the causes of autoimmune diseases such as lupus and rheumatoid arthritis (Moulton & Tsokos, 2015; Sun et al, 2023). So yeah, dysregulated T cell signaling is bad!

Instead, proper substrate selection based on simple electrostatic patterning allows T-cell signaling to turn on in the proper context of actual illness, while remaining off when not needed to prevent autoimmune disorders.

In this article we talked about how protein charge is important for biomolecular interactions and enzyme function. Relatively simple electrostatic principles are used to regulate cellular responses to the surrounding environment and brain function. Furthermore, such simple charged interactions can contribute to diseases like cancer and autoimmune disorders.

Given everything that we’ve discussed in this article, am I sure that protein charge is really, really important? I’m not just sure, I’m positive! (Yes, that’s a joke).

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