Overview: N-terminus and C-terminus
In the field of protein biochemistry, comprehending the fundamental structures of polypeptide chains holds paramount importance. Proteins, comprised of elongated sequences of amino acids, feature distinct terminal ends known as the N-terminus and C-terminus. These terminal points not only mark the inception and conclusion of a protein but also play pivotal roles in its synthesis, configuration, and biological function.
What is the N-terminus and C-terminus?
N-terminus
The N-terminus also referred to as the amino-terminus, NH₂-terminus, or N-terminal end, initiates the polypeptide chain. It is characterized by a free amine group (-NH₂) and represents the initial amino acid in the sequence.
C-terminus
The C-terminus, also known as the carboxyl-terminus, COOH-terminus, or C-terminal end, concludes the polypeptide chain. It is distinguished by a free carboxyl group (-COOH) and marks the final amino acid in the sequence.
What is the primary structure of the N-terminus and C-terminus?
N-terminus
Structurally, the N-terminus commences with an amino acid possessing a free amine group. In eukaryotic proteins, this is typically methionine, encoded by the start codon (AUG). This free amine group is critical for various post-translational modifications that influence protein function and stability.
C-terminus
The C-terminus terminates with an amino acid featuring a free carboxyl group. This terminal carboxyl group is equally crucial for post-translational modifications, such as amidation, which can impact the protein's functional properties and interactions with other molecules.
What is the function of the N-terminus and C-terminus?
N-terminal Targeting Signals
The N-terminus is the foremost segment of the protein to emerge from the ribosome during biosynthesis. It frequently harbors sequences serving as targeting signals, akin to intracellular postal codes, directing the protein to its designated cellular locale. Upon arrival, these signals are typically cleaved by a processing peptidase.
Signal Peptide
The N-terminal signal peptide is recognized by the signal recognition particle (SRP), guiding the protein to the secretory pathway. In eukaryotic cells, synthesis occurs at the rough endoplasmic reticulum (ER), while in prokaryotic cells, proteins are exported across the cell membrane. In chloroplasts, signal peptides direct proteins to the thylakoids.
Fig 1. The general structure of a signal peptide. It is composed of three main parts: (1) N-region- the positive-charged domain. (2) H-region- the hydrophobic core, forming α-helix. (3) C-region- the cleavage site, forming β-sheet. (Hajar et al., 2018)
Mitochondrial Targeting Peptide
The N-terminal mitochondrial targeting peptide (mtTP) facilitates protein import into the mitochondrion.
Chloroplast Targeting Peptide
The N-terminal chloroplast targeting peptide (cpTP) enables protein import into the chloroplast.
C-terminal Retention Signals
While the N-terminus often contains targeting signals, the C-terminus may harbor retention signals for protein sorting. A prevalent endoplasmic reticulum (ER) retention signal is the amino acid sequence -KDEL (or -HDEL), which retains proteins in the ER, preventing entry into the secretory pathway.
N-terminal Modifications
The N-terminus of proteins undergoes diverse post-translational modifications that significantly influence their localization and function. These alterations frequently involve the addition of lipid anchors, facilitating membrane association without the incorporation of a transmembrane domain.
N-Myristoylation
A common N-terminal modification is N-myristoylation, where a myristoyl group, a 14-carbon saturated fatty acid, covalently attaches to the glycine residue at the N-terminus. Proteins undergoing N-myristoylation typically possess a consensus sequence at their N-terminus, serving as a signal for this modification. This lipid anchor critically impacts membrane localization and protein-protein interactions.
N-Acylation
Another notable N-terminal modification is N-acylation, particularly the addition of palmitoyl groups. This modification attaches a 16-carbon palmitoyl group to the N-terminal cysteine residue, enhancing the protein's hydrophobicity and promoting association with cellular membranes. N-acylation is vital for the proper functioning of numerous signaling proteins.
Fig 2. Schematic pattern of the N-myristoylation mechanism catalyzed by NMTs. (a) The synthesis of myristoyl-CoA. (b) Cotranslational modification by N-myristoylation. (c) Posttranslational modification by N-myristoylation. (d) Bi Bi mechanism: The apo-NMT (left) first binds the fatty acid chain of myristoyl-CoA to form the myristoyl-CoA-NMT complex (upper) accompanied by substrate binding pocket exposure. Subsequently, the complex allows a nascent protein to bind (right). Then, the NMT catalyzes N-myristoylpeptide formation through chemical transformation and releases the myristoylpeptide and CoA (lower). (Meng et al., 2020)
C-terminal Modifications
The C-terminus of proteins can be modified posttranslationally, often by the addition of lipid anchors that allow the protein to be inserted into a membrane without having a transmembrane domain.
Prenylation
One form of C-terminal modification is prenylation. During prenylation, a farnesyl or geranylgeranyl isoprenoid membrane anchor is added to a cysteine residue near the C-terminus. Small, membrane-bound G proteins are often modified this way.
GPI Anchors
Another form of C-terminal modification is the addition of a glycosylphosphatidylinositol (GPI) anchor. The GPI anchor is attached to the C-terminus after proteolytic cleavage of a C-terminal propeptide. A prominent example of this type of modification is the prion protein.
Fig 3. General representation of the three-step prenylation pathway of Ras proteins. The prenylation of proteins consists of a three-step processing pathway that involves three different enzymes: (1) The soluble protein farnesyltransferase (PFTase) attaches a farnesyl group to a cysteine near the C-terminus. (2) A membrane-bound protease then cleaves the C-terminal tripeptide. (3) A carboxymethyltransferase finally modifies the C-terminal cysteine, producing a hydrophobic protein that targets the membrane. (Veronica. and D. Distefan., 2017)
Specialized Functions of C-terminal Domains
The C-terminal domain (CTD) of some proteins has specialized functions.
CTD of RNA Polymerase II
The carboxy-terminal domain of RNA polymerase II typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is involved in the initiation of DNA transcription, the capping of the RNA transcript, and attachment to the spliceosome for RNA splicing. Other proteins often bind to this domain to activate polymerase activity.
Similarities Between N-terminus and C-terminus
Both termini share several similarities:
- Structural Definition: Both the N-terminus and C-terminus define the structural boundaries of a protein.
- Post-translational Modifications: Both ends can undergo modifications that significantly impact protein function and stability.
- Sequence Notation: In the conventional notation of peptide sequences, both termini are crucial reference points, with the sequence written from the N-terminus to the C-terminus.
Differences Between N-terminus and C-terminus
Despite their similarities, the N-terminus and C-terminus exhibit distinct differences:
- Functional Roles: The N-terminus is primarily involved in the initiation of protein synthesis and cellular targeting, while the C-terminus is key in terminating protein synthesis and influencing functional interactions.
- Structural Features: The N-terminus has a free amine group, whereas the C-terminus has a free carboxyl group.
- Sequence Position: The N-terminus marks the beginning of the amino acid sequence, while the C-terminus marks the end.
Application of N-terminus and C-terminus
Polypeptide Modifications
Polypeptides can undergo modifications at various positions, including the N-terminus, C-terminus, or within the peptide chain, which are essential for altering the properties and functions of peptides and proteins, thereby making them more suitable for specific applications in biotechnology and pharmaceuticals.
To facilitate the attachment of modification groups, standard functional groups on the polypeptide must be available, such as the N-terminal amine, lysine's amine, cysteine's thiol, serine, threonine, tyrosine's hydroxyl groups, arginine's C=N group, and the C-terminal carboxyl group. Chemically synthesized polypeptides often possess a free amine group at the N-terminus and a free carboxyl group at the C-terminus. However, the unstable free ends of proteins can impact research to a certain extent.
Advantages of Polypeptide Modifications
Modifications at the N-terminus, C-terminus, or within the peptide chain confer several significant benefits:
- Enhancing stability: N-terminal acetylation and C-terminal amidation reduce the overall charge of the peptide, which decreases solubility but increases stability. By eliminating terminal charges, these modifications improve cellular entry and enhance biological activity, making peptides more closely resemble natural proteins and valuable for therapeutic uses.
- Enhancing Biological Activity: The N-terminus or C-terminus can protect peptides from enzymatic degradation, with acetylation preventing aminopeptidase activity and amidation hindering carboxypeptidase action. These modifications stabilize peptides and extend their half-life in the bloodstream, increasing their therapeutic efficacy.
Determining protein sequences is essential for understanding their structure and function. The N-terminus and C-terminus sequences are crucial as they impact stability, localization, and interactions. Accurate sequencing of these regions provides valuable insights, assisting in the study of protein function, new therapeutic approaches, and quality control of biology products.
N-terminal Sequencing Methods
N-terminal sequencing is equally important and can be achieved through various techniques:
- Edman Degradation: This classical method sequentially removes and identifies N-terminal amino acids but requires high protein purity.
- Reverse Transcription PCR (RT-PCR): This method deduces the N-terminal amino acid sequence from the corresponding DNA sequence. Although fast and straightforward, it cannot detect post-translational modifications.
- Mass Spectrometry (MS): Mass spectrometry offers high sensitivity, accuracy, and resolution, making it a preferred method for N-terminal sequencing. It can detect modifications and provide detailed information on the N-terminal sequence.
C-terminal Sequencing Methods
The C-terminus can be sequenced using various techniques:
- Carboxypeptidase Digestion: This enzymatic approach cleaves the C-terminal end, allowing identification of the released amino acids.
- Chemical Cleavage: Chemical reagents target and break peptide bonds near the C-terminus.
- Mass Spectrometry (MS): Proteins are digested into smaller peptides and analyzed by mass spectrometry, with the C-terminal sequence determined by examining specific peptide ions.
By determining the N-terminal and C-terminal sequences, researchers can study protein maturation, localization signals, and interactions. This information is vital for designing peptides and proteins with enhanced therapeutic properties, such as improved stability and targeted delivery.
Fig 4. Workflow for identification and sequencing of N-terminus by mass spectrometry. PGAP is pyroglutamate aminopeptidase; the differential peptide map shows peptide maps of protein digests with and without PGAP treatment. (Malgorzata Monika et al., 2019)
Protein Purification
Affinity chromatography is an effective method for purifying proteins by leveraging specific interactions between the target protein and a ligand attached to a solid matrix. This technique ensures both high purity and yield, making it indispensable in research and industrial applications.
Tags for Purification
Adding tags to proteins simplifies their isolation. Common tags include:
- His-tag (Histidine Tag): Consists of six histidine residues that bind to nickel ions (Ni²⁺) on a resin, facilitating efficient purification.
- MBP-tag (Maltose-Binding Protein Tag): Attaches to amylose resins, aiding in protein isolation.
- CBD (Chitin-Binding Domain): Enables binding to chitin resins for purification.
The His-tag is particularly popular due to its ease of use and effectiveness. When a protein has a His-tag at either terminus, it can be captured with Ni-NTA (nickel-nitrilotriacetic acid) resin. The protein binds to the nickel ions on the resin, while impurities are washed away. The target protein is then eluted with a solution containing imidazole, which competes with the His-tag for binding to the nickel.
Advantages of Affinity Chromatography
Affinity chromatography provides several key benefits:
- High Specificity: The tag-ligand interaction is highly specific, leading to the isolation of the target protein with high purity.
- Scalability: This method can be easily scaled, making it suitable for both laboratory and industrial use.
- Speed: Affinity chromatography is a quick method, enabling the rapid purification of proteins.
Fig 5. Production and purification of recombinant protein scheme, involving (1) insertion of recombinant DNA in host cells and transformation process, (2) cloning process, (3) selection of the host cells containing recombinant DNA, (4) growth of the host cells, (5) upscaling, (6) fusion protein purification through affinity chromatography, (7) recognition of fused protein by the affinity ligand through affinity tag, and (8) elution of the purified fusion protein. (Ana Sofia et al., 2014)
Conclusion
Understanding the N-terminus and C-terminus is fundamental in the field of protein biochemistry. At Creative Proteomics, we leverage this knowledge to explore innovative solutions in biotechnology, enhancing protein function, stability, and interaction capabilities. The distinct roles and structural characteristics of these termini underscore their importance in protein biology, providing a foundation for advanced research and application in various scientific and industrial domains.
References
- Owji, Hajar. "A Comprehensive Review of Signal Peptides: Structure, Roles, and Applications." (2018).
- Yuan, Meng. "N-Myristoylation: From Cell Biology to Translational Medicine." (2020).
- Diaz-Rodriguez, V. and M. D. Distefano. "A-Factor: A Chemical Biology Tool for the Study of Protein Prenylation." (2017).
- Vecchi, Malgorzata Monika et al. "Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation." (2019).
- Pina, Ana Sofia et al. "Affinity Tags in Protein Purification and Peptide Enrichment: An Overview." (2014).
For research use only, not intended for any clinical use.
