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Chapter 25. Proteins

Recommended Article: 【Organic Chemistry】 Organic Chemistry Table of Contents

1. Characteristics

2. Amino acids

3. Protein primary structure

4. Protein secondary structure

5. Protein tertiary structure

6. Protein quaternary structure

7. Methods for determining protein structure

8. Protein denaturation

9. Protein metabolism

a. Composition of living organisms

b. Biopolymer library

1. Characteristics

⑴ Components: C, H, O, N, S

⑵ Functions

① Enzymes in metabolic reactions

② Formation of intracellular and extracellular structures: Comprise half of the body’s weight except water.

③ Muscle contraction

④ Immune function

⑤ Hormones and signaling proteins

⑥ Intracellular signaling

⑦ Transport of substances across membranes

⑧ Energy conversion and storage: 4 kcal/g. Mainly in fetuses

⑨ DNA replication, repair, and recombination

⑩ Transcription, translation

⑪ Protein transport and secretion

⑶ Proteins: Composed of one or more polypeptides

⑷ Polypeptide: Formed by peptide bonds between amino acids

⑸ Peptide bond: Dehydration synthesis reaction between amino group (-NH2) and carboxyl group (-COOH)

Figure 1. Dehydration synthesis of two amino acids

2. Amino Acids

⑴ Total of 20 amino acids: Some include selenocysteine (Sec), making it 21

Table 1. Amino acid symbols

⑵ Structure: Central carbon, amino group, carboxyl group, R group

⑶ All amino acids have the same backbone, but there are 20 different R groups. Others are not used in protein synthesis

① Including minor amino acids like selenocysteine can exceed 20

⑷ Zwitter ions: Act as acids or bases. Equivalence point of Zwitter ion

⑸ Amino acid properties vary with side chains

① Nonpolar amino acids: Ala, Ile, Leu, Met, Phe, Pro, Trp, Val, Gly

② Polar, uncharged amino acids: Asn, Cys, Gln, Ser, Thr, Tyr

③ Positively charged amino acids (+): Arg, His, Lys

④ Negatively charged amino acids (-): Asp, Glu

⑤ Amino acids with -OH group: Ser, Thr, Tyr

○ A phosphate group can be added to the -OH group, which is used in signal transduction.

○ Ser, Thr: Involved in phosphorylation cascade in signaling

○ Tyr: Present in tyrosine kinase receptors in signal transduction.

⑥ Amino acids containing sulfur: Cys, Met. Only Cys forms disulfide bonds

⑦ Amino acids containing phenyl group: Phe, Trp, Tyr

⑧ Phenyl group exhibits 280 nm absorption. Used in UV spectrometer quantification

⑨ Amino acids not used in TCA cycle: Leu, Lys

⑹ Amino acid titration

Table 2. pK_a1, pK_a2, pK_aR, pI, hydrophobic index, and ratios in proteins for each amino acid

3. Protein Primary Structure

⑴ Amino acid sequence

⑵ Planar nature of peptide bonds

① Amide plane: Forms a peptide bond. Involves C, O, N, H between two alpha carbons

② Naming of various bonds in the same plane

○ Φ bond: N - C_α bond

○ ψ bond: C_α - C bond

○ ω bond: C - N bond

○ χ bond: C_α - R (side chain) bond

③ The C=O bond exhibits resonance with the ω bond, resulting in the C=O and C-N bonds having 1.5 bonds each, which prevents rotation and fixes their positions.

Figure 2. Planar nature of peptide bonds

4. Protein Secondary Structure

⑴ The hydrogen bonds between the backbone of amino acids (between H and O) result in a three-dimensional coiling or folding structure of the amino acid sequence.

⑵ Alpha helix

Figure 3. Alpha helix structure

① Hydrogen bonds between C=O of nth amino acid and N-H of n+4th amino acid

② Resilient, right-handed helix

③ Characteristics: Hydrogen bonds are present within amino acids, but not between amino acids

④ Hydrophilic amino acids have attractive and repulsive forces that interfere with the formation of alpha helices.

○ Conclusion: Hydrophobic amino acids mainly form alpha helices

○ Frequently observed in membrane-spanning proteins

⑤ Gly: Side chain R is a hydrogen, preventing proper alpha helix formation due to its small size

⑥ Pro: Forms an imino group, limiting hydrogen bonding → prevents alpha helix formation

⑶ Beta pleated sheet

Figure 4. Beta pleated sheet structure

① Characteristics: In addition to hydrogen bonds between the amino acid backbone, there are also hydrogen bonds between the amino acid side chains.

② Common in fibrous proteins like silk

③ Spider silk combines beta pleated sheet with alpha helix

⑷ Ramachandran plot

① Two-dimensional representation of Phi bond on the x-axis and Psi bond on the y-axis

② Alpha helix is concentrated at (-60°, -60°). Beta pleated sheet is concentrated at (-120°, 120°)

③ Used to assess proper residue positions. Clusters on the plot indicate improper positions

④ Example

Figure 5. Ramachandran plot example

5. Protein Tertiary Structure

⑴ Overall three-dimensional structure of a polypeptide

⑵ Different structures arise due to interactions between R groups. Differences in structure determine properties

⑶ Ionic bonds

⑷ Covalent Bond

⑸ Disulfide Bridge: Contributes to a more stable conformation

① Reducing Agent: Breaks disulfide bridges. Examples: β-mercaptoethanol, DTT (dithiothreitol)

② Disulfide bridge is formed in endoplasmic reticulum for eukaryotic cells.

③ Disulfide bridge is formed by PDI (protein disulfide isomerase) in the cytoplasm for prokaryotic cells.

④ Anfinsen’s Experiment

○ NEM (N-ethylmaleimide): Covalently bonds to the -SH group of cysteine that do not participate in disulfide bonds.

○ DTT (including β-mercaptoethanol): -S-S- → -SH + HS-. Functions as a reducing agent to break disulfide bonds.

○ NEM* → DTT → NEM: Quantifies the initially present SH groups. * indicates radioactive isotope.

○ NEM → DTT → NEM*: Quantifies the initially present -S-S- bonds. * indicates radioactive isotope.

○ Conclusion 1: When denaturing factors are removed, proteins revert to their original structure.

○ Conclusion 2: Disulfide bridges contribute irreversibly to the formation of conformation, requiring precise control.

⑹ Hydrophobic Interaction: Force arising due to the aqueous nature of the biological environment.

⑺ Polar Bonds and Hydrogen Bonds between R Groups

⑻ van der Waals Forces

⑼ Specific interactions can cause misfolding of proteins, necessitating chaperone proteins for proper assembly.

① Example 1: Heat Shock Protein: Prevents protein denaturation due to temperature changes.

6. Protein Quaternary Structure

⑴ Interactions between tertiary structure polypeptides.

⑵ Transthyretin (tetramer), Hemoglobin (α₂β₂), Collagen (triple helix)

7. Methods for Determining Protein Structure

⑴ Determination of Primary Structure

① Method 1: Edman Degradation

○ Binds to the N-terminal of polypeptides in mild alkali to sequentially break down amino acids.

○ Only around 10 amino acids can be identified from the N-terminal.

○ Highly traditional method, currently used by very few.

② Method 2: Peptidase

○ Combination of endopeptidases and exopeptidases to appropriately break down and determine amino acids.

○ Endopeptidases: Pepsin, Trypsin, Chymotrypsin, etc.

○ Exopeptidases: Carboxypeptidases, etc.

○ Mechanism of Peptidases

③ Method 3: Ab Array

④ Method 4: Mass Spectrometry

○ 1^st. Electrophoresis & DNA ladder: Can determine purity or mixture.

○ 2^nd. Trypsin treatment.

○ 3^rd. 1^st mass spectroscopy: Construct MS spectrum.

○ 4^th. 2^nd mass spectroscopy: Construct MS/MS spectrum. Also known as tandem MS or fragmentation.

○ 5^th. Reconstruction through spectrum: In the domain of informatics.

○ These methods are 1^st generation techniques and become more complex with consideration of post-translational modifications.

⑵ Determination of Secondary Structure

① Method 1: Circular Dichroism (CD)

② Method 2: Infrared Spectroscopy: Useful for investigating flexible peptide and protein structures.

③ Method 3: Molecular Dynamics Simulation

⑶ Determination of Tertiary and Quaternary Structures

① Method 1: X-ray Crystallography: About 90% of determinations use this method. Allows inference of 3D atomic coordinates.

② Method 2: NMR: About 9% of determinations use this method.

③ Method 3: Cryo-Electron Microscopy (Cryo-EM)

○ Resolution is low but steadily improving. Useful for large protein complexes like capsids and amyloids.

○ Protein’s 3D structure observed after freezing it at -200 ℃.

8. Protein Denaturation

⑴ Denaturation: Loss of function due to changes in conformation caused by factors like salt concentration, pH, and high temperature.

9. Protein Metabolism

⑴ Protein Degradation

⑵ Protein Synthesis

Input: 2022.04.18 00:26