Post-Translational Modifications: Types, Functions, and Analytical Methods

Proteins serve as the fundamental building blocks of biological processes, with their functional capacity deeply rooted in their three-dimensional architecture. While genetic coding determines the amino acid sequence that provides the blueprint for their final structure, the full range of protein functions is not encoded by DNA alone. Through chemical changes that occur after protein assembly—collectively known as post-translational modifications (PTMs)—cells exponentially expand protein functionality. PTMs can dramatically alter a protein's conformation, its interaction with other proteins, and its subcellular localization, which together control intricate physiological mechanisms.

From a structural perspective, PTMs are critical for modulating protein function and their role in cellular networks. Proteins aren't static entities; they undergo conformational changes in response to various cellular signals. These conformational changes are often mediated by PTMs. For example, the addition of phosphate groups, acetyl groups, or ubiquitin can induce conformational changes that either activate or inhibit enzyme function, alter protein-protein interactions, or signal protein degradation.

Figure 1. Proteins in eukaryotic cells can be edited after translation by a wide variety of reversible and irreversible PTM mechanisms. The structure, stability and function of proteins in the cells can be dynamically altered by these PTMs. (Wang et al., 2014)

Types of Post-Translational Modifications and Their Impact on Protein Structure

PTMs encompass a wide variety of chemical modifications, including phosphorylation, acetylation, ubiquitination, glycosylation, and methylation. Each modification has different structural and functional consequences, affecting charge distribution, hydrophobicity, and overall conformation.

Phosphorylation Dynamics

Phosphorylation involves the attachment of a phosphate group (PO₄²⁻) to serine, threonine, or tyrosine residues, introducing a negative charge that can alter the shape of the protein and its interaction sites. This modification plays an important role in signaling, as seen in kinases—enzymes that rely on phosphorylation-induced structural changes to activate or inhibit downstream targets. Additionally, phosphorylation can occur on histidine and aspartate residues, particularly in prokaryotes, where it regulates bacterial signaling and metabolic pathways. By changing the way a protein folds or interacts with other molecules, phosphorylation fine-tunes many cellular pathways.

Figure 2. Regulatory mechanism of protein phosphorylation. Protein phosphorylation is regulated by antagonistic actions of protein kinases and protein phosphatases. An unphosphorylated protein is converted into a phosphoprotein by a protein kinase and the reversal of this reaction is catalyzed by a protein phosphatase. The phosphorylation of a substrate by a protein kinase is an energy-consuming step that converts ATP into ADP. Dephosphorylation of a phosphoprotein by a protein phosphatase involves hydrolysis of the phosphoester bond, thereby liberating a PO₄³⁻ moiety. (Plattner and Bibb, 2012)

Acetylation's Dual Roles

Acetylation involves the addition of an acetyl group (-COCH₃) to lysine residues, neutralizing their positive charge and altering protein function. This modification is particularly well-known for its role in histone regulation, where it reduces the interaction between histones and negatively charged DNA, leading to a more relaxed chromatin structure and enhanced gene expression.

Beyond histones, acetylation also occurs at the N-terminus of proteins, a process known as N-terminal acetylation. This modification is widespread in eukaryotes and plays a critical role in regulating protein stability, localization, and interactions with other cellular components.

Figure 3. Schematic representation of histone acetylation and deacetylation process. Acetylation of lysine (K) residues on substrate histone protein is carried out by histone acetyltransferases (HAT). In the acetylation reaction, acetyl coenzyme A (here Ac-CoA) is converted into coenzyme A (CoA). Subsequently, acetylated histones impact chromatin's structure, making it open and, as a consequence, activating gene expression. The process is reversible due to the action of histone deacetylases (HDAC), the "erasers" that remove the acetyl group from histone tails. Chromatin remains in its condensed, inactive (closed) form. This blocks transcription machinery from accessing genes' promoter regions and, in turn, represses gene expression. (Miziak et al., 2024)

Ubiquitination—Ubiquitin Tagging Mechanisms

Ubiquitin molecules attached to lysine residues act as cellular "eat-me" signals, marking proteins for degradation. These bulky ubiquitin tags distort the structure of target proteins, directing them to the proteasome for breakdown. However, ubiquitination is not limited to protein degradation. It also plays a versatile role in regulating a wide range of cellular processes. For example, monoubiquitination and polyubiquitination with distinct linkage types (e.g., K48, K63) are involved in DNA repair, endocytosis, and signal transduction pathways. Furthermore, ubiquitin-like proteins, such as SUMO and NEDD8, can modify proteins in a similar fashion, significantly broadening the regulatory scope of ubiquitin-like modifications in cellular function.

Figure 4. The ubiquitination and deubiquitination processes. The ubiquitination process is performed by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E1 enzymes activate ubiquitin and transfer it to E2 enzyme. Then, E2 enzymes interact with E3 enzymes, leading to the transfer of ubiquitin from the E2 enzymes to the specific target proteins. The ubiquitinated proteins can be recognized and degraded by the proteasome. Ubiquitination can be reversed by deubiquitination, which is a process of cleaving ubiquitin from target proteins. Deubiquitination is carried out by a class of deubiquitinases (DUBs). (Zheng et al., 2023)

Glycosylation—Sugar-Coated Structures

Glycosylation involves the attachment of carbohydrate groups and typically occurs in the endoplasmic reticulum (N-linked glycosylation) and Golgi apparatus (O-linked glycosylation), but it also occurs in the cytoplasm and nucleus (O-GlcNAcylation, a type of O-linked glycosylation). It can occur in two primary forms: N-linked glycosylation, where the sugars are attached to asparagine residues, and O-linked glycosylation, where they are attached to serine or threonine residues. These modifications can stabilize proteins, increase solubility, and protect against degradation. In addition, glycosylation plays an important role in protein folding, cellular signaling and immune recognition.

Figure 5. Classification of major manifestations of glycosylation on proteins. GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; GPI, glycosylphosphatidylinositol. (He et al., 2024)

Methylation's Subtle Effects

Methylation modifies lysine or arginine residues by adding one or more methyl groups. In histones, this modification affects chromatin structure and gene regulation, either activating or silencing transcription depending on the site and degree of methylation. In non-histone proteins, methylation can affect interaction surfaces and stability, altering how proteins function within cellular pathways.

In addition to lysine and arginine residues, methylation can also occur on histidine and aspartate residues, although these modifications are less well-studied. Histidine methylation, for example, has been implicated in the regulation of enzyme activity and protein-protein interactions in certain bacterial systems. Furthermore, methylation of non-histone proteins, such as transcription factors and RNA-binding proteins, can influence their stability, localization, and function.

Figure 6. Lysine (K) methylation is a dynamic and reversible post-translational modification (PTM) of proteins. Generally, the lysine ε-amino groups can accept up to three methyl groups, resulting in mono-, di-, or trimethyllysine. Lysine methyltransferases (KMTs) catalyze the addition of methyl groups to substrates, while lysine demethylases (KDMs) remove methyl groups. K, lysine; PTM, post-translational modification; KMTs, lysine methyltransferases; KDMs, lysine methyltransferases. (Han et al., 2019)

PTMs in Cellular Processes

PTMs in Cellular Communication: Driving Signal Transduction Pathways

Signal transduction relies heavily on PTMs to regulate the activation and deactivation of signaling proteins. Phosphorylation is one of the most common PTMs involved in signal transduction. Kinases and phosphatases modulate the activity of signaling proteins by adding or removing phosphate groups. These modifications induce conformational changes that either activate or inhibit protein function, allowing cells to respond to external stimuli.

For example, receptor tyrosine kinases (RTKs) undergo autophosphorylation upon ligand binding, triggering downstream signaling cascades. Structural studies have provided valuable insights into how phosphorylation drives conformational changes that propagate signaling. PTMs such as phosphorylation, acetylation, and ubiquitination can alter the protein-protein interaction landscape within signaling networks, making them key regulators of cellular responses.

Epigenetic Control: Post-Translational Modifications in Gene Regulation

PTMs also play a central role in the regulation of gene expression. Acetylation and methylation of histones, for example, alter the accessibility of DNA by modifying chromatin structure. Acetylated histones result in a more relaxed chromatin structure that promotes gene expression, while methylated histones can either activate or repress transcription, depending on the context.

From a structural perspective, PTMs on histones and transcription factors alter their conformation, allowing or preventing their binding to DNA or other regulatory proteins. These changes can either facilitate or block transcription, depending on the nature of the modification.

Proteostasis and Turnover: PTMs in Protein Degradation Pathways

The ubiquitin-proteasome system is an example of how PTMs control protein turnover. Polyubiquitination targets proteins for proteasomal degradation, thereby maintaining cellular protein homeostasis. Dysregulation of this system has been implicated in diseases such as cancer and neurodegeneration.

Molecular Defense Mechanisms: PTMs in Immune Response Modulation

PTMs modulate immune responses by regulating antigen presentation, cytokine signaling, and immune cell activation. For example, glycosylation of antibodies influences their effector functions and interactions with Fc receptors.

Figure 7. Post-translational modifications (PTMs) within the mammalian cell. This figure illustrates some of the most well-known PTMs and their functions within the cell. PTMs are found throughout the cell from the plasma membrane to the nucleosomes present within the nucleus. PTMs play crucial roles in almost all cellular processes including the cell cycle, degradation, apoptosis, cell signaling, transcription, etc. Different proteins modified by the same PTM will not always yield the same response, demonstrating the diverse functions of PTMs within the cell. (Dunphy et al., 2021)

Beyond their roles in signal transduction, gene regulation, protein degradation, and immune responses, PTMs are also critical in regulating the cell cycle, metabolism, and cytoskeletal dynamics. For example, phosphorylation of cyclin-dependent kinases (CDKs) and their inhibitors (CKIs) plays a central role in cell cycle progression. Similarly, acetylation of metabolic enzymes can modulate their activity in response to cellular energy status, while tubulin acetylation influences microtubule stability and function.

Methods for Studying Post-Translational Modifications in Structural Biology

Post-translational modifications (PTMs) play a critical role in protein function, influencing processes such as signal transduction, stability, and interactions. Understanding PTMs and their effects on protein structure is a central focus of structural biology. Several advanced techniques are used to study PTMs, each providing unique insights into their effects on protein structure and function.

Atomic-Resolution Insights: X-Ray Crystallography for Visualizing PTM-Induced Structural Changes

X-ray crystallography remains a cornerstone technique in structural biology, providing high-resolution three-dimensional structures of proteins. It is particularly effective in identifying the precise location and effect of PTMs on protein conformation. For example, phosphorylated kinases studied by crystallography reveal conformational changes associated with their activation, providing critical insights into the mechanisms underlying signal transduction and regulation. Despite its advantages, crystallography requires protein crystallization, which is not always feasible for highly modified proteins.

Dynamic Structural Analysis: NMR Spectroscopy in Mapping PTM Effects on Protein Conformation

NMR spectroscopy allows researchers to study PTMs in solution, enabling the observation of dynamic processes that are often difficult to capture using static techniques. It is an excellent method for determining how PTMs affect protein structure, flexibility, and dynamics in their native environment. NMR is particularly useful for smaller proteins and fragments, providing atomic-level resolution to elucidate PTM-induced changes in molecular interactions and conformational stability.

High-Resolution Visualization: Cryo-Electron Microscopy for Capturing PTM-Induced Structural Dynamics

Cryo-EM has become a transformative tool in structural biology, particularly for the study of large protein complexes and their PTMs. By preserving proteins in their near-native state without the need for crystallization, cryo-EM enables the visualization of PTM effects at high resolution. This technique is invaluable for identifying the structural roles of PTMs within macromolecular assemblies, such as the regulation of enzyme activity or protein-protein interactions in complex biological systems.

Precision Detection: Mass Spectrometry for Identifying and Quantifying PTMs

Mass spectrometry is a versatile and powerful approach for identifying, characterizing, and quantifying PTMs. Advanced MS techniques, such as tandem mass spectrometry (MS/MS) and isotopic labeling, provide detailed insight into the location and stoichiometry of PTMs. MS-based methods are particularly effective for analyzing crosstalk between multiple modifications and determining how these modifications affect protein interactions and networks. The combination of MS with enrichment strategies enhances the detection of low-abundance PTMs, making it an indispensable tool in proteomics and structural biology.

In Silico PTM Modeling: Computational Approaches for Predicting Structural and Functional Impacts

Computational techniques complement experimental methods by providing predictive insights into PTMs and their functional effects. Molecular dynamics simulations provide detailed models of how PTMs alter protein stability, conformational flexibility, and interaction interfaces. In addition, bioinformatics tools can predict PTM sites based on sequence motifs, structural conservation, and evolutionary data, streamlining the identification of modification hotspots. These approaches are particularly valuable for studying PTMs in proteins where experimental data may be limited.

Multi-Method Synergy: Integrating Structural Techniques for Comprehensive PTM Analysis

To gain a comprehensive understanding of PTMs and their influence on protein structure, researchers often integrate these methods. For example, combining mass spectrometry for PTM identification with cryo-EM or X-ray crystallography for structural analysis provides a holistic view of PTM effects. Computational approaches further enhance this integration by providing predictive frameworks and corroborating experimental results.

By combining these cutting-edge techniques, structural biology continues to uncover the intricate role of PTMs in regulating protein function, facilitating advances in areas such as drug discovery, enzymology, and biomolecular engineering.

Figure 8. Schematic PTM Workflow illustrating some of the common steps associated with sample preparation, PTM enrichment, MS and bioinformatics analysis. (Pascovici et al., 2019)

In addition to X-ray crystallography, NMR spectroscopy, cryo-EM, and mass spectrometry, emerging techniques such as single-molecule fluorescence resonance energy transfer (smFRET) and super-resolution microscopy are becoming increasingly important in PTM research. smFRET allows for the real-time observation of dynamic conformational changes induced by PTMs, while super-resolution microscopy enables the visualization of PTM distribution and dynamics within living cells at nanometer resolution. These techniques complement traditional methods and provide new insights into the spatial and temporal regulation of PTMs.

Case Studies

Case 1: Posttranslational Modifications of Intact Proteins Detected by NMR Spectroscopy: Application to Glycosylation

A new method using NMR spectroscopy allows rapid detection and analysis of post-translational modifications (PTMs) without complex sample preparation. The technique, which can be applied to proteins of any size and without isotopic labeling, uses two-dimensional (2D) NMR experiments such as ¹H-¹³C and ¹H-¹H correlation spectra to identify saccharides and their linkages, providing structural insight that complements mass spectrometry. The method simplifies analysis by denaturing proteins, removing size constraints, and providing random-coil chemical shifts. Modifications are detected by deviations from these random-coil positions, with experiments performed in D₂O to minimize signal interference. The use of [[D₄]-urea for denaturation increases sensitivity while avoiding ionic strength issues.

Tests on glycoproteins from bacteria, fungi, plants and animals revealed distinct PTM fingerprints, particularly in the anomeric region of [¹H, ¹³C]-HSQC spectra. Additional experiments such as TOCSY identified complex glycans, including mannans in yeast invertase and various linkages in human serum albumin. The method detected less common modifications, such as rare phosphorylation, and revealed novel PTMs in well-studied proteins, including N-glycosylation in human tumor necrosis factor (TNF). This versatile NMR-based approach enables comprehensive PTM analysis, thereby enhancing the structural understanding of proteins in various biological systems.

Figure 9. Glycosylation detected in denatured bacterial, fungal plant and mammalian proteins using 2D [¹H, ¹H]-TOCSY spectra (120 ms mixing time). The spectral regions show the chemical shift range of the anomeric H1 proton (ω₂) and correlated proton signals. a) in vitro glycosylated bacterial protein AtaC1866–2428 displays correlations to all chemical shifts of the glucose spin system. b) Bromelain displays the previously reported chemical shifts; an additional resonance of unknown origin is indicated by a hash sign at ω₂ = 5.4 ppm. c) Spectrum of human serum albumin, the observed signals mainly correspond to signals of a complex biantennary structure with two sialic acid residues. d) Invertase from S. cerevisae; the observed signals mainly correspond to signals of isolated mannans from Candida glabrata; e) Human TNF expressed in HEK cells. (Schubert et al., 2015)

Case 2: Epigenetic mechanisms to propagate histone acetylation by p300/CBP

The study explores the mechanism by which p300/CBP enzymes read and write histone acetylation to regulate gene transcription. Using cryogenic electron microscopy (cryo-EM), the researchers revealed structures of a p300/CBP multidomain monomer that recognizes acetylated histone H4 N-terminal tails (H4NTac) and acetylates non-H4 histone N-terminal tails (NTs) within the same nucleosome.

Figure 10. a Structure of p300BRPH bound to H2BNT and acetylated H4NT delineated by cryogenic electron microscopy (cryo-EM). Left, top view; right, side view. p300BRPH binds to H4acNuc in a Slinky-like bent conformation via bromodomain and HAT. b Overall structure of p300H2B with H4-di-acetylated nucleosome in cartoon presentation. Color code: orange, p300 bromodomain (BD); cyan, p300 RING and PHD zinc-fingers (RP); magenta, p300 histone acetyltransferase domain (HAT); green, K12/K16-acetylated H4; red, H2B. (Kikuchi et al., 2023)

In summary, post-translational modifications are central to protein functionality and cellular regulation, with profound implications for structural biology. By altering protein conformation, stability, and interactions, PTMs enable the dynamic regulation of biological processes. Advances in structural biology techniques have greatly improved our understanding of PTMs and revealed their roles in health and disease. Continued exploration of PTMs will provide deeper insights into their mechanisms and facilitate the development of targeted therapies, underscoring their importance in biomedical research.

Figure 11. The structure of a typical glycoprotein, human EPO. PDB code: 1BUY.

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