Proteomics Chapter 1 PDF

# Proteomics ## Chapter 1: Introduction to proteomics ### Proteomics: - A rapid tool to identify proteins. - Maps the interactions of proteins in their context. ### Introduction to Amino Acids and Proteins: **1.1. Amino Acids** - An organic compound. - Has a side chain. **1.2. Essential Ami...

# Proteomics ## Chapter 1: Introduction to proteomics ### Proteomics: - A rapid tool to identify proteins. - Maps the interactions of proteins in their context. ### Introduction to Amino Acids and Proteins: **1.1. Amino Acids** - An organic compound. - Has a side chain. **1.2. Essential Amino Acids:** - Cannot be made by the body. - His, Iso, Leu, Lys, Met, Phe, Thr, Trp, Val **1.3. Non-Essential Amino Acids:** - Ala, Arg, Asp, Asn, Glu, Gly, Pro, Ser, Tyr, Gln, Cys **1.4. Asymmetric Carbon:** - Chiral atoms ### Classification: - **Aromatic:** Phe, Trp, Tyr - **Basic:** Lys, Arg, His - **Acidic:** Asp, Glu - **Hydrophobic:** Ala, Iso, Leu, Met, Phe, Trp, Tyr, Val - **Hydrophilic:** Asp, Glu, Arg, Gln, Asn, Lys - **Glycine Titration** ### Some Definitions: - **Peptide:** A chain of amino acids (AA) ranging from 2 to 50 AA. - **Dipeptide:** 2 AA. - **Tripeptide:** 3 AA. - **Apoprotein:** Contains just AA. It is an inactive form of protein. - **Holoprotein:** Functional form of a protein. ## Chapter 2: Protein identification ## Chapter 3: Challenges in proteomics (enjeux) utilization ## Chapter 4: Introduction to proteomics ### Proteomics: - A rapid tool to identify protein and map their interactions in context. ### Introduction to Amino Acids and Protein: **1.1. Amino Acids** - An organic compound. - It's an organic compound. - It's an organic compound. - It's an organic compound. - It's an organic compound. - It's an organic compound. - NH₂ -C - COOH C. Terminus - R - Side chain **1.2. Essential A.A.:** - Can't be made by the body. - His (H), Iso (I), Leu (L) - Lys (K), Met (M), Phe (F) - Thr (T), Trp(W), Val (V) **1.3. Non-Essential:** - Ala (A), Arg (R), Asp.(D) - Asn (N), Glu (E), Gly (G) - Pro (P), Ser(S), Tyr(Y) - Gln (Q), Cys (C). **1.4. Asymmetric Carbon** - Chiral atoms - All are chiral except Glycine ### Classification : - **Aromatic:** Phe, Trp, Tyr - **Basic:** lys, Arg, His - **Acidic:** Asp, Glu - **Hydrophobic:** Ala, Iso, Leu, Met, Phe, Trp, Tyr, Val - **Lydrophilic:** Asp, Glu, Arg, Gln, Asn, Lys - **Glycine Titration** ### Some difinitions: - **Peptide:** chain of AA from 2 to 50 A.A - **Di peptide:** 2AA - **Tripeptide:** 3AA - **Apoprotein:** contien just AA and it's an inach ve form of proteine and these proteins may become funclianal and called holoprotein ## Secondary : - Refers to coiling and folding of peptides. - **Formation of hydrogen bonds:** Between amino acids by the H and O of the COO and NH in the principal chain. **α-helix:** - 3.6 (13) - **Number of AA:** in one helix - **Φ:** -57° - **ψ:** -47° - **Pitch:** 5.4 nm - **Rise:** 5.4 - **3.6 AA turn:** - **Notice:** Ser, Gly, Pro, and Tyr are important A.A to know. **β-Sheets:** - **Number of Atoms:** N H C - C O - **H bonds are:** - **Parallel:** unit - **Anti-parallel:** H bonds are perpendicular to the sheet - **No elasticity:** - **More stable:** Due to steric repulsion - **Cys-pep:** C - S - S - C - **J I helix:** 4.46 ## Tertiary Structure: - **3D arrangement of Secondary structures** - **Bonds between side chains:** we have more interactions - **Ionic bond:** - Between anion and cation. - Θ Θ - **Non-interaction:** (repulsion) - **Interaction** - By hydrophobic bonds: between water hating A.A. ## Types of Motifs Structures: - **Super-secondary structure of protein:** formed by alpha helix and B sheets by loops and turns. **1. Helix-turn-helix motif:** **2. Helix-loop-helix:** ## Heteroprotein: - It's multiple polypeptides chain, often of different types (another non-protection groups). ## Protein Domains: - Independent folding Unit of the tertiary structure. - Formed by disulfide and ionic bonds. - **Des motifs:** (sub-units) - Each can have a specific function and can interact with other protein domains. - **Binding domains:** - They work together to realize one function. ## Dynamic Protein: - **Ex of RCPG (Receptor coupled à la protéine G):** A transmembrane protein containing α-helix. - **State of protein G (receptor):** - **Active:** when protein G is present - **Inactive:** when protein G is not present - **External signal:** Activates the protein G - **Domain:** - Contains 50-250 amino acids - Three types of Domains - Structural - Binding/regulation - Oligomerization - **Quaternary structure:** - Contains sub-units - (Domain = sub-unit) - **Hemoglobin:** stabilized by disulfide bridge. - Hydrophobic amino acids are important to stabilize it. ## Protein Folding : - **DNA -> RNA -> peptid** - **After Synthesis:** we have an inactive protein (infolded). - **Protein will fold** to have the functional structure. - **Infolded protein:** - Can be activated by an effector molecule. - **Active proteins:** - Can interact with non-proteic partners. - **Cofactor:** - Non proteic group - Interacts with infolded proteins to give native protein. - **Binding Site:** Non native protein can bond with cofactor -> native protein ## Prosthetic Groups: - Non protein part: irons, Mg2+, methyl, glucose, lipids - **Holoprotein:** Metalloprotein, glycoprotein, lipoprotein - **(Part protein + non protein = apoprotein)**. ## Experiment of Anfinsen: - **Urea:** A substance that breaks down non-covalent bonds. - **β-mercaptoethanol:** Breaks disulfide bonds. - **RNAse:** 124 AA. - **Experiment:** - When using these substances, the protein will be denatured due to breaking down of bonds. - By removing β-mercaptoethanol the disulfide bonds are recovered. ## The recovered structure of ARNase - **Experiment of Lernthal:** - **Paradox:** highlights the contradiction between the large number of possible confirmations a protein could adopt and the rapidity of which a protein folds into their biological structure. - **To calculate the number of max possible and stable conformation:** - **m**: Number of amino acids. - **m²:** Number of confirmation possible. - **Angular:** An angle turns confirmation, for each sequence of amino acids there are 2 possibilities of angular conformation. - **m = 2.** - **Ex:** 100 AA with 99 peptide bonds. - There are 198 ψ and Φ. - **Number of stable confirmations:** - The time to calculate the time for adopting a confirmation is 10s. - So 10⁹⁹ x 10¹³s = 1.267 x 10¹¹². ## Funnelshaped Energy Landscape: - **Represents the energy landscape that a protein navigates to achieve a native structure during a folding process.** **Wide opening:** The top of the funnel, land space is large and represents a wide range of confirmations (pluss. confirmations. - **It's the infolded state.** **Narrowing funnel:** The energy land space narrows into a funnel shape - **This narrowing indicates fewer energetically favorable confirmations.** - **(كلما زادت التعريجات يعني نقص الطاقة يعني أقل طاقة ملي قبلها وكلما ) ** ## Deep Well: - **At the bottom of the funnels is the native state.** - **It's characterized by the lowest energy level.** - **The path of folding is not random.** It's specific and must have pathways to achieve 3D Structure that has other molecules to help this structure to do the folding. ## The Proteines Chaperon: - **Infolded protein is trapped** - **Protein:** - Due to disruptions of protein chaperons that can lead protein to misfold. - Due to environmental factors. - **Misfolding:** - Other proteins chaperon lead the protein to fold correctly. - **Amorphous amyloid aggregates:** Fibrils, aggregates ## Defective Protein Folding: - **Aggregates are not eliminated:** They can cause dysfunction of normal cells. - **Toxicities:** Can be toxic to cells, leads to cell damage or death. - **Toxicity is particularly evident in neurons:** Can cause neurogenerative diseases such as Alzheimer, Parkinson and Huntington. - **Inflammation:** Can cause an immune response. - **Impaired Cellular Processes:** Impaired function of cells. ## Heat Shock Protein: - **Hsp (nb):** (paid molecular) - **HPS 70:** - Thrale - **Protein folding and refolking of misfolded proteins.** - **Protect the protein after leaving ribosome.** - Assist on the folding of newly synthesized protein. - **Disagregation and refolking of aggregates.** ## Chaperonine Family: - **HPS 60:** - **A large family assisting in protein folding within a protected environment (ex: cavite).** - **Refolking of denatured protein.** - **Prevention of agregation by encapsulating these protein.** - **Role of encapsulation of protein (acts as a molecular cage).** ## Co Chaperonin: - **HPS 40:** - **Works with Hsp to stimulate ATPase activity to hydrolyse ATP** - **Facilitates the folding of newly synthesized protein and helps to ensure a functional protein correctly folded.** ## Protein Denaturation: - **The process when a protein loses it's function structure (3D structure) due to factors extern.** - **Factors extern:** - **Heat:** - **pH (Acids/Bases)** - **Solvents:** - **High pressure:** - **Ultra sound:** - **Physical Agents:** - **Acids and bases:** Highly acidic or highly alkaline. - **High Pressure:** - **Ultra sound:** - **Organic Solvents:** Like alcohol and acetone can break hydrophobic interactions and hydrogen bonds. - **Detergents:** Distrupt the hydrophobic interactions. - **Reducing agents:** Like mercaptoethanol can break disulfide bonds. - **Physical Agents:** - **Heat (high T):** Can distrupt the weak bonds. - **Radiations:** X-rays, UV can break chemical bonds of protein. - **Physical action:** Shaking and mechanical agitation can break non-covalent bonds. ## Post-Translational Modifications (PTM): - **Chemical modifications that happen to proteins after they are synthesized** - **Important for proper function:** - Regulation - Activation - Signalization - Localization - **The role of PTM:** - **Differentiation** - **Protein Degradation** - **Signalization and regulation** - **Regulatory of gene expression** - **Protein-protein interactions** - **Common post-translational modifications:** **1. Protein Phosphorylation:** - Phosphate (PO₄)^3- is added to protein molecule. - By the eng Protein Kinase. **2. Acetylation:** - Addition of acetyl group (CO-CH₃). - By the eng Histone-Acetyl-transferase. - **Can occur on the Amino group (N-terminal) of lysine.** - **Important for:** - Gene regulation and localization. - Stability of protein and localization. **3. Protein Amidation:** - Add an NH₂. - Involves the conversion of the carboxy-lique (C-terminal) to amide group by added NH₂ (-CONH₂). - **It's a manar to protect protein.** - **It can make protein less sensitive to proteolytic degradation.** **4. Protein Ubiquitination** - **A small protein called ubiquitin can be attached to a protein.** - **The process is carried out by a series of enzymes:** - **E1:** Ubiquitin activating - **E2:** Ubiquitin conjugating - **E3:** Ubiquitin ligase. - **They work together to transfer the ubiquitin molecule to a target of protein** **5. Protein Glycosylation:** - **Attachement of N-glycosamine on the oxygen of side chain of Thr and Ser on the protein.** - **GLCNAC:** - According to glu - Avaibility - **Source of N-glycosamine** - The hexamine biosytheme: - **Glutamate > F-6-p > Glucosamine 6-p > UDP-N-Acetyl glycosamine** - **This process has a role in:** - Regulation - Control of protein stability and activity - **Dysfunction of this process can cause diabetes of type 2.** ## Cleavage: - **The process of breaking down a protein into smaller peptides (fragments).** - **Example:** - Modification of preproinsulin. - Preproinsulin is the precursor form of insulin. - It undergoes several modifications before becoming functional. - **The steps of this process:** **1. Signal peptide sequence cleavage:** - Preproinsulin has a signal peptide sequence. - This sequence is cleaved. **2. Disulfide bond formation:** - Proinsulin contains disulfide bridge to ensure its stability. **3. C-chain removal:** - Proinsulin contains C chain, which is located between A chain and B chain. - The C chain is cleaved. - This step gives us the mature insulin. ## Study of Protein Function: - **Study of protein structure:** - **Sequencing** (for primary structure) - **Sequencing:** - It is a process determining the order of AA in a peptide chain. - **Two Methods:** - **Edman degradation:** Removes and identifies the amino acids from the protein chain. - **Mass spectrometry:** Scientists analyze the mass spectrum and can determine the exact mass of the protein-deducting AA sequence of peptide. - **For study secondary structure:** - **Circular Dichroism:** - This technique measures the differential absorption of left-handed polarized light vs right-handed polarized light by optically active molecules. - **Non-polarized light:** - **Polarized light** - **Right waves:** - **We need monochromator that polarizes the light waves:** - **Monochromate:** - **Polariser:** - **The left-handed circularly polarized light and the right handed circularly polarized light passes throughout the sample.** - **Secondary structures don't absorb in equally way the left-handed light and the right-handed light.** - **This technique determines α helices, ß-sheets, random coils.** - **Wave length (nm):** - **For Study 3D structure:** - **X-ray crystallography:** - **Method used on crystalline substance:** - **Crystallization of protein:** The protein is purified then mixed with various crystallization reagents to find suitable conditions to be crystallized (pH, Tm, precipitants). - **X-ray diffraction:** - X-rays are diffracted by the atoms of crystals. - The heavier atoms are more efficient for the X-rays diffraction. This resulting a diffraction pattern, this pattern contains information of arrangements of atoms in crystals of protein. - **Electron density map:** - The pattern is captured on detection and subjected to mathematical analysis (by complex math algorithm). - It converts the pattern into electron density map (electron distribution). - Used to know atom localisation and build structural model - **NMR** (Nuclear Magnetic Resonance): - Analyzes protein structure interactions, dynamics at an atomic resolution in various sample states.