Test: Proteins- 2 - NEET PG MCQ

• Hb electrophoresis: Hb S migrates more slowly in an electric field because it is less negatively charged due to the replacement of glutamate by valine.
• Sickling Test
• HPLC is used to fractionate Hb.
• Isoelectric Focusing
• An allele-specific oligonucleotide probe identifies the haemoglobin (Hb) S allele.

The immunochemical method employed for the identification of antigens and antibodies serves qualitative purposes:

• Passive Gel Diffusion: This involves a concentration gradient for a single reactant, where the antigen diffuses into agar that is impregnated with antibody.
• Single Immuno Diffusion: Radial Immune Diffusion is a quantitative approach based on Single Immuno Diffusion.
• Double Diffusion (Ouchterlony Technique): A concentration gradient is created for both the antigen and antibody. This allows for the comparison of two or more test materials, providing a straightforward method to determine if the antigens in the test specimens are identical, cross-reactive, or non-identical. This technique is named after the Swedish physicist Orjan Ouchterlony.
• Immuno-electrophoresis: This technique is utilised to separate and identify various protein species within a sample. It is particularly useful for studying antigen mixtures and human gammopathies.

Western Blotting is also a significant method in this context.

Native gel electrophoresis relies on charge.

Ultrafiltration and ultracentrifugation are dependent on density and molecular mass.

2D electrophoresis is determined by isoelectric pH and molecular weight (size).

Gel filtration chromatography, also known as molecular sieve chromatography, is based on molecular weight and size.

• SDS-PAGE provides an equal negative charge, effectively masking the natural charge of the protein.
• Currently, proteins are separated solely based on molecular weight (size) using SDS-PAGE combined with 2-mercaptoethanol or dithiothreitol.
• These agents oxidatively cleave disulfide bonds, allowing for the separation of multimeric protein components.

• FRAP stands for Fluorescence Recovery After Photobleaching.
• This technique utilises fluorescent dyes that emit coloured light when illuminated.
• However, if exposed to very high-intensity light, these dyes cannot fluoresce, a phenomenon known as photobleaching.
• Subsequently, the dyes can recover their fluorescence.

Methods of Protein Sequencing

Sanger's Technique

• Sanger's Reagent is 1, Fluoro 2, 4 Dinitrobenzene°.
• This reagent derivatises the amino terminal residues.
• The first protein sequenced using this method was insulin by Fredrick Sanger.
• He received the Nobel Prize in 1958.
• Only dipeptides or tripeptides can be sequenced with this technique.

Edman's Technique

• This method employs Edman's Reagent (Phenyl Iso-Thio-cyanate).
• Phenyl Isothiocyanate derivatises the amino terminal of the polypeptide.
• Edman's Technique can sequence multiple residues (5-30 residues) from a single polypeptide sample, unlike Sanger's method.

Quadrupole mass spectrometers are typically employed to measure the masses of molecules weighing 4000 Da or below. In contrast, time-of-flight mass spectrometers are utilised to ascertain the greater masses of entire proteins.

Types of Collagen

• Major collagen found in bone: Type I (90%)
• Major collagen in the dermis, ligaments, and tendons: Type I (80%)
• Major collagen present in cartilage: Type II (40-50%)
• Major collagen in hypertrophic cartilage: Type X
• Major collagen in the aorta: Type I and Type III (20-40% each)
• Major collagen in the basement membrane: Type IV
• Most abundant collagen: Type I
• Major collagen in keloids: Type III

Type I collagen in wound healing:

• Initially, a provisional matrix is formed, consisting of fibrin, plasma fibronectin, and type III collagen.
• This is subsequently replaced by a matrix primarily made up of type I collagen.

• Glycine-X-Y repeats: Every third amino acid in collagen is a glycine.
• Alpha Chain: The polyproline helix consists of three residues per turn, twisted in a left-handed manner.
• Each polypeptide chain is made up of 1000 amino acids.
• Triple helical structure: Three of these alpha chains are coiled into a right-handed super helix.
• Quarter Staggered arrangement:
  • Lateral association of the triple helical units occurs.
  • Each unit is offset longitudinally from its neighbour by just under one-quarter of its length.
• This arrangement contributes to the tensile strength of collagen fibres.

The greater the number of disulfide bonds, the more resilient the protein becomes. Keratin contains a high concentration of cysteine.

Proteins are categorised based on their shape into:

• Fibrous Proteins
  • Elongated, needle-like, long cylindrical, or rod-shaped
  • Minimal solubility in water
  • Regular secondary structure
  • Axial ratio > 10
  • Examples include collagen, elastin, and keratin
• Globular Proteins
  • Spherical, oval, or spheroidal in shape
  • Easily soluble in water
  • Axial ratio <>
  • These proteins carry out dynamic functions, such as albumin, globulin, and most enzymes

Triple helix structure in collagen

• Quarter staggered arrangement found in collagen
• Covalent cross-links present in collagen
• Desmosine cross-links occurring in elastin

Distribution of collagen:

• Type I is found in non-cartilaginous connective tissues such as bone and tendon.
• Type II is present in cartilage and the vitreous humour.
• Type IV is mainly found in the basement membrane, with Type XIX being rare.
• Type VII is located at the dermal-epidermal junction.
• Type III is associated with extensible connective tissues like skin, lungs, and the vascular system.
• Type X is found in hypertrophic cartilage.
• Type XVII is present in skin hemidesmoses.
• Type XIX is associated with rhabdomyosarcoma cells.
• Type XXV is located in the brain.
• Type XXVI is found in the testis and ovary.

• Function of Golgi bodies: O-Glycosylation of proteins
• Protein sorting
• Processing of oligosaccharide chains in glycoproteins

SRP-R, the signal recognition particle receptor, is a docking protein. The Translocon (Sec 61 complex) is made up of three membrane proteins, forming the protein-conducting channel within the ER. The signal sequence emerges from the ribosome and attaches to the SRP, which temporarily halts the elongation of the polypeptide chain (known as elongation arrest).

• Both SRP and SRP-R bind to GTP, not ATP.
• The SRP is released from SRP-R, allowing translation to continue.

The signal hypothesis suggests that the protein is embedded in the ER membrane concurrently as its mRNA is being translated on polyribosomes, a process referred to as cotranslational insertion.

• Principal Components involved in Endoplasmic Reticulum Translocation:
• N-terminal signal peptide
• Polyribosomes
• SRP, signal recognition particle
• SR, signal recognition particle receptor
• Sec 61, the translocon
• Signal peptidase
• Associated proteins (e.g., TRAM and TRAP)

TRAM, or Translocating Chain Associated Membrane Protein, facilitates the translocation of specific proteins. The role of TRAP, or Translocon-associated Protein Complex, remains unclear.

• N-linked glycoproteins undergo glycosylation in the RER.
• The RER, along with the Golgi apparatus, is integral to the exocytic pathway of protein sorting.

Inclusion (I) cell disease is characterised by:

• A disorder that affects lysosomal enzymes.
• The recognition marker for these enzymes is mannose 6 phosphate.
• The absence of the Golgi-located N-acetyl glucosamine phosphotransferase.

A variety of proteins have the amino acid sequence KDEL (Lys-Asp-Glu-Leu) at their carboxyl terminal. These KDEL-containing proteins:

• Initially travel to the GA in COPII transport vesicles.
• Interact with a specific KDEL receptor protein, allowing for temporary retention.
• Subsequently return to the ER in COPI transport vesicles, where they dissociate from the receptor and are retrieved.

HOEL sequences (where H represents histidine) perform a similar function. These processes lead to the net localisation of certain soluble proteins within the ER lumen. The SEC61 gene encodes a channel that facilitates the passage of secretory proteins under construction into the endoplasmic reticulum lumen.