Protein Structure, Purification, and Function: Study Notes for Biochemistry

Protein Structure and Homology

Levels of Protein Structure

• Primary Structure: The linear sequence of amino acids in a polypeptide, connected by peptide bonds. Determines the protein's function and three-dimensional structure.

• Secondary Structure: Local, ordered folding patterns of the polypeptide backbone, stabilized mainly by hydrogen bonds between backbone amide (NH) and carbonyl (C=O) groups. Main types: alpha helices and beta sheets.

• Tertiary Structure: The overall three-dimensional shape of a single polypeptide chain, including all its secondary structures and side chain interactions.

• Quaternary Structure: The arrangement and interaction of multiple polypeptide subunits in a protein complex.

Protein Homology and Sequence Conservation

• Homologous Proteins: Proteins that are evolutionarily related and share similar sequences or structures.

• Paralogs: Homologous proteins within the same species.

• Orthologs: Homologous proteins in different species.

• Invariant Residues: Amino acids that remain unchanged across all homologs, often crucial for protein function (e.g., active sites).

• Variable Residues: Positions in the sequence that differ among homologs.

• Conservative Substitutions: Replacement of an amino acid with a similar one (e.g., leucine for isoleucine), usually preserving protein structure and function.

Protein Purification Techniques

Column Chromatography

• Separates proteins based on physical or chemical properties using a stationary phase (beads/matrix) and a mobile phase (buffer with proteins).

• Types of chromatography:

  • Ion Exchange: Separates by charge.

  • Size Exclusion (SEC): Separates by size; large proteins elute first, small proteins elute later.

  • Affinity: Separates by specific binding to ligands attached to the matrix.

  • Hydrophobic Interaction: Separates by hydrophobicity.

• Protein detection in fractions:

  • Absorbance at 280 nm: Aromatic amino acids (Trp, Tyr, Phe) absorb UV light; used to detect protein presence.

  • BCA Assay: Detects peptide bonds via copper reduction, forming a purple complex measured at 562 nm. Less dependent on aromatic content.

SDS-PAGE and Electrophoresis

• SDS-PAGE: Denatures proteins and coats them with negative charge, allowing separation by size only. Smaller proteins migrate further.

• Reducing Agents: DTT or beta-mercaptoethanol break disulfide bonds for complete denaturation.

• Coomassie Blue Stain: Used to visualize protein bands after electrophoresis.

Size-Exclusion Chromatography (SEC)

• Separates proteins based on size; maintains quaternary structure, allowing estimation of native protein complex size.

• Band broadening can reduce resolution over time due to diffusion.

Estimating Molecular Weight

• SEC: Calibrate with proteins of known molecular weight, plot log(MW) vs. elution volume (Ve).

• SDS-PAGE: Use a molecular weight ladder, plot MW vs. migration distance (Rf).

• Comparison of SEC and SDS-PAGE can reveal subunit composition (e.g., tetramers, heteromers).

Ion-Exchange Chromatography

• Separates proteins by charge using charged beads (DEAE for anion exchange, CM for cation exchange).

• Elution by increasing salt concentration or changing pH.

Affinity Chromatography

• Uses specific ligand-protein interactions (e.g., antibody-antigen, His-tag/Ni-NTA).

• Elution by adding free ligand, changing pH, or increasing salt.

• His6 Tag: Six histidines engineered into protein bind Ni2+ on NTA resin; eluted with imidazole.

Sequential Purification Steps

1. Crude extract (cell lysis)

2. Ammonium sulfate precipitation (fractionation by solubility)

3. Ion-exchange chromatography

4. Size-exclusion chromatography

5. Affinity chromatography (if applicable)

• Other precipitation methods: acetone (denaturing), trichloroacetic acid (TCA, harsh, denaturing).

• Centrifugal filters concentrate proteins by size exclusion.

Isoelectric Focusing and 2D Electrophoresis

• Isoelectric Focusing: Separates proteins by isoelectric point (pI) in a pH gradient gel.

• 2D Electrophoresis: First dimension: isoelectric focusing (by pI); second dimension: SDS-PAGE (by size).

Protein Sequencing Methods

Chemical Sequencing Steps

1. Separate polypeptide chains (reduce disulfide bonds with DTT or beta-mercaptoethanol).

2. Identify N-terminal residue (FDNB/Sanger's reagent or dansyl chloride).

3. Identify C-terminal residue (carboxypeptidase digestion).

4. Cleave protein into smaller peptides (trypsin, chymotrypsin, cyanogen bromide).

5. Sequence fragments and reconstruct full sequence using overlapping fragments.

N-terminal and C-terminal Determination

• Dansyl Chloride: Labels free amino groups; after hydrolysis, fluorescent tag identifies N-terminal residue.

• FDNB (Sanger's Reagent): Labels N-terminal amino group; after hydrolysis, identifies first residue.

• Edman Degradation: Sequentially removes and identifies N-terminal amino acids using PITC, up to 30-50 residues.

• Carboxypeptidases: Enzymes that remove C-terminal residues; different types have different specificities.

Endopeptidases and Chemical Cleavage

• Trypsin: Cleaves after Lys or Arg.

• Chymotrypsin: Cleaves after aromatic residues (Phe, Trp, Tyr).

• Cyanogen Bromide: Cleaves after Met, converting it to homoserine lactone.

• Pepsin: Cleaves after aromatic residues, active at acidic pH.

Mass Spectrometry-Based Sequencing

• Peptides are ionized and separated by mass-to-charge ratio (MS1).

• Selected ions are fragmented (collision cell), producing b-ions (N-terminus) and y-ions (C-terminus).

• MS2 spectrum provides sequence information based on fragment masses.

Peptide Bonding and Protein Backbone

Peptide Bond Properties

• Peptide C-N bond has partial double-bond character due to resonance, making it rigid and planar.

• Rotation is restricted around the peptide bond (omega, usually 180° trans).

• Rotation occurs around phi (N–Cα) and psi (Cα–C) angles, determining backbone conformation.

Ramachandran Plot

• Plots allowed phi (x-axis) and psi (y-axis) angles for amino acids in proteins.

• Shows favored regions for secondary structures (e.g., beta sheets, alpha helices).

• Glycine is more flexible and occupies more regions; proline is more restricted.

Protein Secondary Structure

Alpha Helix

• Right-handed coil stabilized by hydrogen bonds between C=O of residue i and N-H of residue i+4.

• 3.6 residues per turn; rise per residue = 1.5 Å; pitch per turn = 5.4 Å.

• R-groups project outward, minimizing steric clashes.

• Helix dipole: N-terminus is partially positive, C-terminus is partially negative.

• Stabilized by intrahelical hydrogen bonds, salt bridges, and hydrophobic interactions.

• Destabilized by proline (helix breaker, rigid, no amide H) and glycine (too flexible).

Beta Sheet

• Extended zigzag conformation; beta strands align side by side, stabilized by interstrand hydrogen bonds.

• Antiparallel sheets (opposite directions) are more stable than parallel sheets (same direction).

• Beta turns connect strands, often containing glycine and proline.

Factors Affecting Alpha Helix Stability

• Electrostatic interactions between charged R groups.

• Bulkiness of adjacent R groups.

• Interactions between R groups 3-4 residues apart (hydrophobic, salt bridges, H-bonds).

• Presence of proline or glycine.

• Helix dipole interactions at termini.

• Van der Waals interactions in the core.

Protein Structure Determination

X-Ray Crystallography

• Proteins are crystallized; X-rays diffract through the crystal, producing a pattern that reveals electron density.

• Fourier transform converts diffraction data into a 3D electron density map.

• Atomic positions are modeled into the density; provides a static, high-resolution structure.

Supersecondary Structure and Protein Classes

Supersecondary Structures (Motifs)

• Stable combinations of secondary structure elements (e.g., beta-alpha-beta loop, alpha-alpha corner, beta barrel).

• Motifs recur in many proteins and are associated with specific functions.

Globular Proteins

• Compact, water-soluble proteins with hydrophilic residues on the surface and hydrophobic residues inside.

• Examples: myoglobin, hemoglobin.

Protein Denaturation

• Loss of structural integrity (except primary structure) due to heat, pH, solvents, or chaotropic agents (urea, guanidinium chloride).

• Denaturation disrupts function but does not break peptide bonds.

Protein Classes

• Enzymes (catalysts), regulatory proteins, transport proteins (e.g., hemoglobin), storage proteins (e.g., ferritin), contractile/motile proteins (actin, myosin), structural proteins (collagen, keratin), scaffold proteins, protective proteins (immunoglobulins).

Protein-Ligand Binding and Oxygen Transport

Ligand Binding

• Ligand: Molecule reversibly bound to a protein via non-covalent interactions.

• Binding Site: Region on protein complementary to ligand in size, shape, and chemical properties.

• Induced Fit: Ligand binding induces conformational change, enhancing complementarity.

Oxygen-Binding Proteins: Myoglobin and Hemoglobin

• Oxygen is poorly soluble in water; myoglobin and hemoglobin evolved to bind and transport O2 efficiently.

• Heme (Protoporphyrin IX + Fe2+): Prosthetic group that binds O2; iron must be in ferrous (Fe2+) state.

• Heme is buried in protein, coordinated by a proximal histidine (His F8) and binds O2 at the other axial position.

• Distal histidine (His E7) stabilizes O2 binding and reduces CO binding affinity.

Binding Equilibria and Affinity

• Association constant: $K_a = \frac{[PL]}{[P][L]}$

• Fractional saturation: $\theta = \frac{[L]}{[L] + K_d}$

• Dissociation constant: $K_d = \frac{1}{K_a}$; lower $K_d$ means higher affinity.

• For O2 binding: $\theta = \frac{pO_2}{pO_2 + P_{50}}$

Myoglobin vs. Hemoglobin

• Myoglobin: Monomeric, binds one O2, hyperbolic binding curve, high affinity (good for storage, not transport).

• Hemoglobin: Tetramer (2 alpha, 2 beta), binds four O2, sigmoidal binding curve (cooperative binding), affinity varies with pO2 (good for transport).

Hemoglobin Structure and Allostery

• T State (Tense): Deoxy form, stabilized by salt bridges, low O2 affinity.

• R State (Relaxed): O2-bound form, higher O2 affinity, subunit interactions change upon O2 binding.

• O2 binding triggers T to R transition, increasing affinity in other subunits (cooperativity).

Cooperative Binding and the Hill Equation

• Cooperative binding: $\theta = \frac{[L]^n}{[L]^n + K_d^n}$

• Hill equation: $\log\left(\frac{\theta}{1-\theta}\right) = n \log pO_2 - n \log P_{50}$

• Hill coefficient (n): n > 1 indicates positive cooperativity; for hemoglobin, n ≈ 2.8.

Allosteric Regulation of Hemoglobin

• 2,3-Bisphosphoglycerate (BPG): Binds to central cavity, stabilizes T state, decreases O2 affinity.

• pH (Bohr Effect): Lower pH (higher [H+]) stabilizes T state, promotes O2 release; higher pH stabilizes R state, increases O2 affinity.

• CO2 Binding: CO2 forms carbamate at N-terminus, stabilizing T state and promoting O2 release.

Sickle Cell Anemia

• Caused by a single amino acid substitution (Glu → Val) at position 6 of beta chain.

• Valine creates a hydrophobic patch, leading to hemoglobin aggregation and sickle-shaped RBCs.

• Results in blocked capillaries, reduced O2 delivery, and pain episodes.

Tables

Summary Table: Protein Purification Methods

Method	Principle	Key Feature	When Used
Ion Exchange	Charge	Separates by net charge at given pH	Early/mid purification
Size Exclusion (SEC)	Size	Large proteins elute first	After partial purification
Affinity	Specific binding	High specificity, high purity	Final purification
Ammonium Sulfate Precipitation	Solubility	Fractionates by salt-induced precipitation	Initial fractionation
SDS-PAGE	Size (denatured)	Analytical, not preparative	Assess purity/size

Summary Table: Endopeptidase Specificity

Enzyme/Reagent	Cleavage Site
Trypsin	After Lys (K) or Arg (R)
Chymotrypsin	After Phe (F), Trp (W), Tyr (Y)
Cyanogen Bromide	After Met (M)
Pepsin	After aromatic residues (F, Y, W)

Summary Table: Hemoglobin Allosteric Effectors

Effector	Effect on Hb	Mechanism
BPG	Decreases O2 affinity	Stabilizes T state
H+ (low pH)	Decreases O2 affinity	Bohr effect, stabilizes T state
CO2	Decreases O2 affinity	Carbamate formation, stabilizes T state

Additional info: Academic context and explanations have been expanded for clarity and completeness. Tables have been inferred and summarized for study purposes.