Control of protein activity by photoinduced spin polarized charge reorganization

This study demonstrates that photoinduced, spin-polarized charge redistribution within proteins, triggered by site-specific ruthenium photosensitizers and modulated by circularly polarized light, acts as an electrical allosteric signal that significantly alters protein binding affinity and enzymatic activity.

The Gist

Imagine a protein as a complex, squishy machine inside a living cell. Usually, we think of these machines working based on their shape—like a key fitting into a lock. But this paper suggests there's another way to control them: by using electricity and spin, much like flipping a switch or changing the magnetic orientation of a gear.

Here is the story of what the scientists discovered, broken down into simple concepts:

1. The Setup: A Protein with a "Light Switch"

The researchers took a specific protein called PGK (which acts like a tiny factory worker, moving parts around to help cells make energy). They attached a special "photosensitizer" to it. Think of this photosensitizer as a solar-powered battery glued to the protein.

When they shine a light on this battery, it doesn't just get warm; it shoots an electric charge (an electron or a "hole") into the protein. This is like plugging a wire into a machine and suddenly sending a surge of electricity through its internal wiring.

2. The Discovery: Light Changes How the Protein Behaves

When they turned on the light, two surprising things happened:

• The "Handshake" Got Stronger: The protein became much better at grabbing onto a specific antibody (like a magnet getting stronger). The binding happened twice as fast when the light was on.
• The "Factory" Slowed Down: The protein's main job (making energy) actually slowed down by three times when the light was on.

It's as if shining a light on a car engine made the engine run slower, but made the car's door latch snap shut much faster.

3. The Twist: It Only Works with "Left-Handed" Light

This is the most magical part. The researchers tried shining different types of light:

• Straight light: Worked a little.
• Right-handed spinning light: Did nothing.
• Left-handed spinning light: Worked perfectly.

Why? The scientists believe the protein acts like a chiral (handed) filter. Because the protein is twisted like a spiral staircase, it only lets electrons with a specific "spin" (a quantum property, like a tiny top spinning clockwise or counter-clockwise) pass through. The left-handed light creates the right kind of spinning electrons to get through the protein's "gate." If the light is the wrong "handedness," the electrons bounce off or don't get injected, and nothing happens.

4. Location Matters: Where You Plug In Counts

The effect depended entirely on where they glued the light-sensing battery onto the protein.

• When the battery was near the "handshake" spot, the handshake got stronger.
• When the battery was near the "factory floor" (the active site), the factory slowed down.
• If they moved the battery to a spot far away from these areas, the light had almost no effect.

This proves that the electricity isn't just heating the protein up; it's traveling through the protein's internal wires to change how specific parts of the machine behave.

The Big Picture

The paper claims that electricity and charge movement are a hidden language proteins use to control themselves. Just as a conductor uses a baton to tell an orchestra to play louder or softer, a sudden shift in electric charge inside a protein can tell it to work faster, slower, or stick to things more tightly.

Crucially, this isn't just about static electricity (like a balloon stuck to a wall); it's about moving charges and their spin. The researchers showed that by using a specific type of spinning light, they could remotely control a protein's behavior, proving that "charge reorganization" is a real, powerful way nature (and potentially us) can tune biological machines.

Technical Summary

Technical Summary: Control of Protein Activity by Photoinduced Spin Polarized Charge Reorganization

Problem and Context
While the role of bioelectricity at the organismal level and the importance of static electrostatics in protein functions (such as association kinetics and enzymatic catalysis) are well-established, the dynamic response of proteins to external electric fields remains poorly understood. Specifically, the potential contribution of protein polarizability and charge reorganization to biological function is not fully appreciated. Existing models often rely on pre-organized, static charge distributions, neglecting the possibility that external fields or internal charge motions could act as allosteric signals. This study addresses the gap in understanding how phototriggered charge injection and subsequent charge reorganization within a protein influence its function, particularly in the context of the Chiral Induced Spin Selectivity (CISS) effect.

Methodology
The researchers employed a site-specific labeling strategy on Phosphoglycerate Kinase (PGK), a 415-residue enzyme.

• Protein Engineering: A ruthenium photosensitizer (Ru), capable of injecting electrons or holes, was covalently attached to specific cysteine residues introduced via mutagenesis. Two primary mutant constructs were used: Q9C (Ru attached near the C-terminal His-tag, ~10 Å away) and S290C (Ru attached near the ATP binding site, ~55 Å from the C-terminus).
• Surface Immobilization:
  • Protein-Protein Association: Ru-PGK was immobilized on gold or glass surfaces via a His-tag. Anti-His antibodies labeled with Alexa 647 were introduced to monitor binding kinetics.
  • Enzymatic Activity: Ru-PGK was attached to supported lipid bilayers on glass to allow continuous illumination during catalytic assays.
• Illumination Conditions: Experiments were conducted under dark conditions, linearly polarized (LP) light, left circularly polarized (LCP) light, and right circularly polarized (RCP) light (470 nm laser).
• Readout:
  • Binding: Total Internal Reflection Fluorescence Microscopy (TIRFM) was used to count individual antibody-antigen association events over time.
  • Enzymatic Activity: A coupled assay monitored the conversion of NADH to NAD+ (via 3-phosphoglycerate to 1,3-bisphosphoglycerate conversion) by measuring absorbance changes at 340 nm.

Key Results

1. Modulation of Protein-Protein Association:

   • Illumination significantly enhanced the binding rate of anti-His antibodies to the C-terminal His-tag of PGK.
   • At 2 seconds, LP illumination increased the association rate by a factor of 2.25 ± 0.05 compared to the dark.
   • Spin Selectivity: This enhancement was observed only with LCP light; RCP light and LP light (in the context of spin-specificity comparisons) did not produce the same effect, indicating that the charge reorganization is spin-polarized.
   • Position Dependence: When the photosensitizer was moved to position 290 (farther from the C-terminus), the illumination effect on binding was negligible, demonstrating that the effect is highly dependent on the spatial relationship between the charge injection site and the interaction interface.

2. Modulation of Enzymatic Activity:

   • Illumination suppressed the enzymatic activity of PGK.
   • With Ru at position 290 (near the active site), enzymatic turnover decreased by a factor of 3.3 ± 0.2 under illumination. This suppression occurred with both LP and LCP light but not RCP.
   • With Ru at position 9, the suppression was smaller (factor of 1.8 ± 1) but still significant.
   • The effect was reversible; turning off the light restored the reaction rate. No effect was observed in the absence of the Ru photosensitizer.

3. Spin Polarization Mechanism:

   • The differential response to LCP versus RCP light confirms that the electrons involved in the observed dynamics are spin-polarized. This is attributed to the CISS effect, where the chiral structure of the protein acts as a spin filter, allowing preferential injection of a specific spin state depending on the light polarization.

Key Contributions

• Direct Demonstration of Charge Reorganization: The study provides direct experimental evidence that photoinduced charge injection can alter protein function, establishing charge reorganization as a viable mechanism for allosteric regulation.
• Spin-Dependent Allostery: The work links protein function modulation to electron spin polarization, showing that the chiral nature of proteins can filter spin states to control biological activity.
• Positional Sensitivity: The results highlight that the magnitude and direction of the functional change depend critically on the location of the charge injection relative to the functional site (active site or binding interface).

Significance
The paper claims to establish a new role for electrical polarization and charge reorganization in modulating protein activities, complementing known mechanisms like conformational changes. The findings suggest that:

• Electric fields and charge motions, rather than just static charges, play a dynamic role in regulating biological activity at the molecular level.
• The CISS effect allows for the control of protein function via the spin state of injected charges, offering a novel pathway for photo-controlling bioactivity without relying solely on conformational changes induced by photoexcitation.
• These mechanisms may be relevant in cellular environments where electric fields are abundant, particularly near membranes, potentially affecting membrane proteins and membrane-interacting proteins.

The authors conclude that future work using phototriggered charge injection could delineate specific charge rearrangement pathways within proteins and further define their impact on protein function, potentially leading to new methods for generating photo-controlled enzymes and sensors.