Interferon Regulatory Factor 3, commonly known as IRF3, is a protein that plays a significant role in the body’s innate immune system. It functions as a transcription factor, meaning it helps turn specific genes on or off, particularly those involved in responding to viral infections. IRF3 is typically found in an inactive state within the cytoplasm of cells, awaiting signals to initiate its protective functions, which include the production of type I interferons.
Predicted Versus Observed Molecular Weight
The predicted molecular weight of a protein is calculated directly from its amino acid sequence. For human IRF3, which consists of 427 amino acids, this calculated weight is approximately 47 kilodaltons (kDa). This value represents the protein’s mass in its most basic, unmodified form.
However, when researchers observe IRF3 using common laboratory techniques like SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), its observed molecular weight often appears slightly higher. This experimentally determined size typically falls within the range of 50-55 kDa, even when the protein is not yet fully activated. This discrepancy between the predicted and observed weights hints at inherent properties or subtle modifications of the protein in a cellular environment.
The Role of Post-Translational Modifications
A primary reason for the variation in IRF3’s observed molecular weight, particularly its increase upon activation, involves post-translational modifications (PTMs). These are chemical changes that occur to a protein after it has been synthesized, affecting its structure and function. Phosphorylation, the addition of phosphate groups to specific amino acid residues, is a significant PTM for IRF3.
Upon sensing a viral infection, cellular kinases like TBK1 and IKBKE become activated and phosphorylate IRF3 on multiple serine and threonine residues, predominantly in its C-terminal domain. Specific sites include Serine 385, Serine 386, and Serine 396. This addition of negatively charged phosphate groups not only increases the protein’s mass but also induces a conformational change, altering how IRF3 interacts with the SDS detergent used in electrophoresis. These changes cause the phosphorylated IRF3 to migrate more slowly through the gel, appearing as a higher molecular weight band on a Western blot, indicating its active state.
Beyond phosphorylation, IRF3 can also undergo ubiquitination, another PTM involving ubiquitin attachment. Different types of ubiquitin linkages can influence IRF3’s stability or DNA binding. For instance, K48-linked ubiquitination often targets IRF3 for degradation by the proteasome, which can lead to an increase in its apparent molecular weight.
Dimerization and Complex Formation
Following phosphorylation, IRF3 undergoes a structural change that promotes its self-association, forming a homodimer. This dimerization is a necessary step for IRF3 to translocate into the nucleus and activate interferon gene transcription. The formation of this dimer involves interactions between the phosphorylated serine residues on one IRF3 monomer and positively charged regions on another.
Standard SDS-PAGE uses denaturing conditions that break apart these protein-protein interactions. On such a gel, even if IRF3 has formed dimers in the cell, individual IRF3 monomers (both phosphorylated and unphosphorylated) are typically observed. To visualize the full dimeric complex, which would have an approximate molecular weight of 100-110 kDa, researchers must employ non-denaturing or native PAGE techniques, which preserve protein-protein interactions.
Experimental Determination and Considerations
The standard laboratory technique for observing IRF3’s molecular weight and its modifications is SDS-PAGE followed by Western blotting. This method separates proteins by size, allowing researchers to distinguish between different forms of IRF3. When performing SDS-PAGE, selecting the appropriate percentage of acrylamide gel is important for resolving the distinct forms of IRF3.
A 7.5% acrylamide gel is recommended for achieving high resolution and separating the unphosphorylated IRF3 from its slower-migrating, hyperphosphorylated forms. For detection, researchers typically use specific antibodies. “Total IRF3” antibodies recognize all forms of the protein, providing an overall picture of IRF3 levels. To specifically identify the activated state, “phospho-specific” antibodies are used to pinpoint the shifted band corresponding to phosphorylated IRF3.