Biochemistry: Proteins - Their Primary Structure and Biological Functions

Fundamental Architecture and Hierarchical Organization of Proteins

• Classification by Shape and Solubility:

  • Fibrous Proteins: Possess relatively simple, regular, linear structures. They typically play structural and mechanical roles within cells and tissues and are predominantly water-insoluble (for example, collagen).

  • Globular Proteins: Tightly folded into compact, roughly spherical conformations. Their polypeptide chains fold such that hydrophobic side chains are buried within the interior core while hydrophilic residues are exposed to the surrounding aqueous environment, rendering them water-soluble (for example, myoglobin).

  • Membrane Proteins: Reside in direct association with cellular lipid bilayer membranes. They fold with hydrophobic amino acid side chains exposed across their membrane-spanning segments to contact the fatty acyl tails of bilayer phospholipids, whereas hydrophilic residues project into aqueous extracellular or cytosolic regions (for example, bacteriorhodopsin).

• Four Hierarchical Levels of Protein Structure:

  • Primary Structure ($1^\circ$): The unique, linear covalent sequence of amino acid residues joined by peptide bonds. Example: Bovine pancreatic ribonuclease A contains exactly $124$ amino acid residues, lacks tryptophan ($\text{Trp}$) entirely, and contains four stabilizing covalent disulfide bridges ($\text{Cys}26-\text{Cys}84$, $\text{Cys}40-\text{Cys}95$, $\text{Cys}58-\text{Cys}110$, and $\text{Cys}65-\text{Cys}72$).

• Secondary Structure ($2^\circ$): Local, periodic spatial arrangements of the polypeptide backbone stabilized by hydrogen bonds between the peptide amide ($\text{N}-\text{H}$) and carbonyl ($\text{C}=\text{O}$) groups:

  • $\alpha$-Helix: A rod-like spiral formed as the $N-C_\alpha-C$ backbone winds around an imaginary central longitudinal axis. Depicted symbolically as an undulating ribbon or a solid cylinder.

  • $\beta$-Pleated Strand: An extended zigzag backbone conformation where consecutive amide planes are oriented perpendicular to the sheet plane, and side-chain substituents ($\text{C}_\beta$) alternate projecting above and below the strand. Depicted symbolically as a broad arrow pointing from the N-terminus to the C-terminus.

• Tertiary Structure ($3^\circ$): The overall three-dimensional folding of a single polypeptide chain into a compact native architecture. Tertiary structure is represented via distinct structural abstractions:

  • Backbone trace: Depicts the spatial trajectory of the polypeptide chain backbone.

  • Backbone plus side chains: Includes all covalent bonds and amino acid functional groups.

  • Ribbon diagram: Emphasizes secondary structural elements ($\alpha$-helices and $\beta$-strands) and intervening loop segments.

  • Space-filling model: Displays all atoms as van der Waals spheres, illustrating packing density, surface clefts, and total molecular volume.

• Quaternary Structure ($4^\circ$): The noncovalent or covalent subunit organization and spatial packing of oligomeric proteins consisting of two or more distinct polypeptide chains. Example: Hemoglobin exists as an $\alpha_2\beta_2$ tetramer comprising two identical $\alpha$-chains and two identical $\beta$-chains, each coordinating a prosthetic heme group.

• Distinction Between Conformation and Configuration:

  • Configuration: The geometric arrangement of atoms in a molecule that is fixed by covalent bonds. Conversion between alternative configurations requires the breaking and reforming of covalent chemical bonds (such as stereoisomeric inversion between $\text{D}$-glyceraldehyde and $\text{L}$-glyceraldehyde).

  • Conformation: The spatial array achieved by a molecule purely through rotation about single covalent bonds without the rupture of any covalent linkages (such as interconversion among staggered and eclipsed rotamers of 1,2-dichloroethane).

  • In proteins, the peptide bond ($\text{C}-\text{N}$) has partial double-bond character and is planar, preventing free rotation. However, two of every three bonds along the backbone—the $\text{N}-\text{C}_\alpha$ single bond (torsion angle $\phi$) and the $\text{C}_\alpha-\text{C}$ single bond (torsion angle $\psi$)—are freely rotatable, providing expansive conformational freedom.

Isolation, Fractionation, and Purification of Cellular Proteins

• Physicochemical Principles of Protein Purification:

  • Cellular lysates contain thousands of distinct proteins that are separated on the basis of differences in size, shape, electrical charge, and biological binding affinity.

  • Isoelectric Point ($pI$) and Solubility: A protein displays minimum solubility at its isoelectric point ($pI$), where the net molecular charge is zero ($\text{net charge} = 0$). Under this condition, electrostatic repulsion between adjacent protein molecules is minimized, promoting self-association and precipitation.

  • At $\text{pH} < pI$, a protein acquires a net positive charge; at $\text{pH} > pI$, it acquires a net negative charge.

  • Salting-In: At low ionic strengths ($1\,\text{mM}$ to $20\,\text{mM}$), the addition of neutral salts increases protein solubility by providing counterions that shield localized opposite charges on the protein surface, decreasing intermolecular ionic aggregation.

  • Salting-Out: At elevated ionic strengths (for example, $4\,\text{M}$ ammonium sulfate), hydrated salt ions compete with the protein for water molecules of solvation. As water becomes structured around the salt ions, the hydration shell shielding the protein is stripped away, exposing hydrophobic patches and inducing rapid aggregation and precipitation.

• Colorimetric Estimation of Protein Concentration:

  • Bicinchoninic Acid (BCA) Assay: Relies on the alkaline biuret reduction of cupric ions ($\text{Cu}^{2+}$) to cuprous ions ($\text{Cu}^+$) by protein peptide backbones.

  • Cuprous ions ($\text{Cu}^+$) are coordinated by two molecules of bicinchoninic acid to yield an intensely purple chelate complex ($\text{BCA}-\text{Cu}^+$) with an optical absorbance maximum at $562\,\text{nm}$, directly proportional to protein concentration.

• Quantitative Metrics in a Purification Protocol:

  • Specific Activity: Defined as the total units of enzyme activity divided by the total mass of protein in milligrams:   $\text{Specific Activity} = \frac{\text{Total Activity}}{\text{Total Protein}}\,(\text{units/mg})$

  • Specific activity increases as an enzyme is progressively enriched relative to total contaminating protein.

  • Representative Five-Step Purification Scheme:

  • Step 1 (Crude extract): Volume = $3800\,\text{mL}$; Total Protein = $22,800\,\text{mg}$; Total Activity = $2460\,\text{units}$; Specific Activity = $0.108\,\text{units/mg}$; Percent Recovery = $100\%$.

  • Step 2 (Salt precipitate): Volume = $165\,\text{mL}$; Total Protein = $2800\,\text{mg}$; Total Activity = $1190\,\text{units}$; Specific Activity = $0.425\,\text{units/mg}$; Percent Recovery = $48\%$.

  • Step 3 (Ion exchange chromatography): Volume = $65\,\text{mL}$; Total Protein = $100\,\text{mg}$; Total Activity = $720\,\text{units}$; Specific Activity = $7.2\,\text{units/mg}$; Percent Recovery = $29\%$.

  • Step 4 (Molecular sieve chromatography): Volume = $40\,\text{mL}$; Total Protein = $14.5\,\text{mg}$; Total Activity = $555\,\text{units}$; Specific Activity = $38.3\,\text{units/mg}$; Percent Recovery = $23\%$.

  • Step 5 (Immunoaffinity chromatography): Volume = $6\,\text{mL}$; Total Protein = $1.8\,\text{mg}$; Total Activity = $275\,\text{units}$; Specific Activity = $152\,\text{units/mg}$; Percent Recovery = $11\%$.

  • Net outcome: Specific activity increases from $0.108\,\text{units/mg}$ to $152\,\text{units/mg}$, achieving a greater than $1400$-fold overall purification.

• Chromatographic and Electrophoretic Fractionation Techniques:

  • Dialysis: Macromolecular solutions are retained inside a semipermeable membrane bag immersed in a large volume of buffer (dialysate). Small solutes and ions diffuse down their chemical potential gradients until equilibrium is attained across the membrane, while large macromolecules remain inside.

• Ion-Exchange Chromatography (IEC): Separates molecules on the basis of net surface charge. In cation-exchange chromatography, the solid matrix consists of negatively charged resin beads. Negatively charged solutes pass through unhindered, whereas positively charged molecules bind to the beads and are subsequently eluted in discrete fractions using increasing salt concentrations or pH shifts.

• Gel Filtration (Size-Exclusion / Molecular Sieve) Chromatography: Separates molecules strictly according to Stokes radius and molecular mass. The column is packed with porous polymer beads:

  • Large molecules exceeding the pore inclusion threshold are excluded and migrate only through interstitial volume, eluting first at the void volume ($V_o$; $V_e \cong V_o$).

  • Smaller molecules penetrate the internal aqueous labyrinth of the beads and follow an extended path, eluting later at elution volume $V_e$ approaching the total column volume ($V_t$).

• Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE): Resolves denatured polypeptides according to molecular weight. Sodium dodecyl sulfate ($\text{CH}_3(\text{CH}_2)_{11}\text{OSO}_3^-\text{Na}^+$) binds to polypeptides at a constant stoichiometric ratio of approximately $1.4\,\text{g}$ SDS per $1.0\,\text{g}$ protein (one SDS molecule per two amino acid residues). This confers a uniform negative charge that dwarfs intrinsic protein charge. Sieving through cross-linked polyacrylamide generates a linear relationship between relative electrophoretic mobility and the logarithm of molecular weight ($\log(\text{MW})$).

• Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE): Resolves complex mixtures using two orthogonal parameters:

  • First Dimension (Isoelectric Focusing, IEF): Polypeptides migrate through a gel containing a continuous pH gradient (pH $4$ to pH $10$) until each species arrives at the exact pH matching its isoelectric point ($pI$), where net molecular charge becomes zero and migration halts.

  • Second Dimension (SDS-PAGE): The IEF tube or strip gel is placed on top of an SDS-polyacrylamide slab and electrophoresed perpendicularly, fractionating species based on molecular weight.

• Affinity Chromatography: Exploits biological specificity. Ligands (metabolites, cofactors, inhibitors, or antibodies) are covalently attached to an insoluble column matrix (such as agarose, polyacrylamide, or cellulose). When a cell extract is applied, only the specific target protein binds to the ligand, while all non-target proteins pass through unadsorbed. The bound protein is eluted by adding high concentrations of free ligand. Single-step purifications of $1000$-fold or greater are achieved routinely.

Primary Structure Determination and Chemical Sequencing Methodologies

• Historical Sequencing of Insulin:

  • Frederick Sanger in 1953 sequenced the two polypeptide chains of bovine insulin, demonstrating that every molecule of a given protein possesses an invariant, ordered sequence of amino acids.

  • Bovine insulin consists of an A chain ($21$ amino acid residues, containing an intrachain disulfide bond between $\text{Cys}6$ and $\text{Cys}11$) and a B chain ($30$ amino acid residues), covalently linked by two interchain disulfide bonds ($\text{A}7-\text{B}7$ and $\text{A}20-\text{B}19$).

• The Classical Six-Step Sequencing Strategy:

  • Step 1: Subunit Separation and Purification: Subunits associated via noncovalent interactions are dissociated using denaturants: extreme pH, $8\,\text{M}$ urea, $6\,\text{M}$ guanidine hydrochloride ($\text{guanidine}\cdot\text{HCl}$), or high salt concentrations (e.g., ammonium sulfate).

  • Step 2: Cleavage of Disulfide Bridges:

  • Oxidative cleavage: Treatment with performic acid ($\text{HCOOOH}$) converts cystine residues irreversibly into cysteic acid residues ($-\text{SO}_3^-$).

• Reductive cleavage: Disulfides are reduced using sulfhydryl reagents such as $\beta$-mercaptoethanol ($\text{BME}$), dithiothreitol ($\text{DTT}$), or dithioerythritol ($\text{DTE}$). To prevent re-oxidation, reduced free thiols are permanently capped via alkylation using iodoacetic acid ($\text{ICH}_2\text{COOH}$) to produce $S$-carboxymethylcysteine derivatives, or $3$-bromopropylamine ($\text{BrCH}_2\text{CH}_2\text{CH}_2\text{NH}_2$).

• Step 3: Identification of N-Terminal and C-Terminal Residues:

  • N-terminal analysis: Reaction with Edman's reagent (phenylisothiocyanate, PITC) selectively labels the free $\alpha$-amino group under mildly basic conditions. Anhydrous acid cleavage releases a cyclic phenylthiohydantoin (PTH) amino acid derivative without hydrolyzing remaining peptide bonds.

  • C-terminal analysis: Exopeptidase digestion with Carboxypeptidase A (which hydrolyzes any C-terminal residue except Pro, Arg, and Lys) or Carboxypeptidase B from hog pancreas (which hydrolyzes exclusively C-terminal Arg and Lys residues).

• Step 4: Fragmentation of Polypeptide Chains: Enzymatic or chemical cleavage is used to fragment chains into smaller peptides, which are sequenced.

• Step 5: Alternative Fragmentation: Step 4 is repeated with an agent of distinct cleavage specificity to create an independent set of overlapping peptide fragments.

• Step 6: Reconstruction of the Complete Sequence: Overlapping sequences of the two peptide libraries are aligned to deduce the uninterrupted primary sequence.

  • Cleavage Specificities of Proteases and Chemical Reagents:

• Trypsin: Cleaves peptide bonds on the carboxyl (C) side of positively charged basic residues, arginine ($\text{Arg}$) and lysine ($\text{Lys}$), provided the following amino acid is not proline ($\text{Pro}$). Digests yield peptides terminating in C-terminal Arg or Lys, plus a single peptide derived from the authentic C-terminus.

• Chymotrypsin: Cleaves on the C-side of aromatic residues: phenylalanine ($\text{Phe}$), tryptophan ($\text{Trp}$), and tyrosine ($\text{Tyr}$), as well as leucine ($\text{Leu}$); blocked if proline follows.

• Clostripain: Cleaves on the C-side of arginine ($\text{Arg}$) residues ($\text{Arg} \gg \text{Lys}$).

• Staphylococcal Protease: In phosphate buffer ($\text{pH } 7.8$), cleaves on the C-side of aspartate ($\text{Asp}$) and glutamate ($\text{Glu}$); in ammonium bicarbonate or acetate buffer ($\text{pH } 4.0$), it is specific for glutamate ($\text{Glu}$).

• Endopeptidase Lys-C: Cleaves specifically on the C-side of lysine ($\text{Lys}$).

• Cyanogen Bromide ($\text{CNBr}$): Chemically cleaves peptide bonds on the C-side of methionine ($\text{Met}$) residues in $70\%$ formic acid ($\text{HCOOH}$):

  • Step 1: Nucleophilic attack of the Met thioether sulfur atom on the carbon of cyanogen bromide ($\text{Br}-\text{C}\equiv\text{N}$) with displacement of $\text{Br}^-$.

  • Step 2: Nucleophilic attack by the Met carbonyl oxygen on the side-chain $\gamma$-carbon with displacement of methyl thiocyanate ($\text{H}_3\text{C}-\text{S}-\text{C}\equiv\text{N}$), generating a cyclic iminolactone intermediate.

  • Step 3: Aqueous hydrolysis cleaves the peptide backbone at the Met position, leaving a peptide terminating in a C-terminal homoserine lactone.

• Hydroxylamine ($\text{NH}_2\text{OH}$): Chemically cleaves asparaginyl-glycyl ($\text{Asn}-\text{Gly}$) peptide bonds.

• Dilute Acid Hydrolysis ($\text{pH } 2.5, 40^\circ\text{C}$): Cleaves aspartyl-prolyl ($\text{Asp}-\text{Pro}$) peptide bonds.

  • Worked Sequence Reconstruction Example:

• Trypsin cleavage of an unknown peptide produces: A-E-F-S-G-I-T-P-K and L-V-G-K.

• Staphylococcal protease cleavage produces: F-S-G-I-T-P-K and L-V-G-K-A-E.

• Overlap alignment:

  • L-V-G-K matches the N-terminus of L-V-G-K-A-E.

  • A-E at the C-terminus of L-V-G-K-A-E overlaps with the N-terminus of A-E-F-S-G-I-T-P-K.

  • Deduced sequence: L-V-G-K-A-E-F-S-G-I-T-P-K.

  • Sequence Analysis of Catrocollastatin-C:

• Catrocollastatin-C is a $23.6\,\text{kDa}$ hemorrhagic metalloproteinase ($216$ amino acids) found in the venom of the western diamondback rattlesnake (Crotalus atrox).

• The uninterrupted sequence was assembled by aligning:

  • Automated Edman degradation of the intact N-terminus (residues $1-42$).

  • Cyanogen bromide proteolytic fragments ($\text{M1}$ through $\text{M5}$).

  • Endopeptidase Lys-C fragments ($\text{K3}$ through $\text{K6}$).

  • Staphylococcal protease digestion fragments ($\text{E13}$ through $\text{E15}$).

Mass Spectrometry for Molecular Weight Determination and De Novo Sequencing

• Principles of Biological Mass Spectrometry:

  • Mass spectrometry (MS) measures the mass-to-charge ratio ($m/z$) of gas-phase protein ions.

  • Electrospray Ionization (ESI-MS): A liquid protein solution is sprayed through a fine glass capillary maintained under high electrical potential against a drying gas countercurrent. Desolvated microdroplets evaporate, generating intact, multiply charged cations ($[M + zH]^{z+}$).

  • A typical $20\,\text{kDa}$ protein picks up $10$ to $30$ protons.

  • The resulting mass spectrum reveals a series of sharp peaks whose adjacent $m/z$ values differ by the mass and charge of a single proton ($1\,\text{Da}$, $+1$ charge). Decreasing $m/z$ values denote increasing charge states ($z$).

• In aerolysin K, charge states range from $30+$ ($m/z \approx 1579$) to greater than $50+$ ($m/z \approx 948$), deconvoluting to an intact molecular mass of $47,342\,\text{Da}$.

• Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS): Samples are embedded in an ultraviolet-absorbing crystalline organic matrix (e.g., sinapinic acid). Laser pulses vaporize the matrix into a microplasma, transferring energy and ionizing proteins into predominantly singly charged ($[M + H]^+$) gas-phase ions that are resolved by flight velocity down an evacuated drift tube.

  • Tandem Mass Spectrometry (MS/MS) and Collision-Induced Dissociation:

• Instrumentation consists of two mass spectrometers positioned in series around an intervening collision cell:

  • $\text{MS-1}$: Scans and isolates a single precursor peptide ion of interest from a complex digest.

  • Collision Cell: The precursor ion undergoes collision-induced dissociation (CID) through collisions with inert gas molecules (helium, $\text{He}$, or nitrogen, $\text{N}_2$), fragmenting primarily at peptide backbone amide bonds.

  • $\text{MS-2}$: Resolves the resulting daughter fragment ions to identify sequence-specific mass differences.

• Step-by-Step De Novo Sequencing of a Tryptic Peptide:

  • In a tryptic digest, fragmentation creates a series of C-terminal daughter ions ($y$-type ions) carrying a charge at the C-terminal basic residue. Subtracting the mass of adjacent peaks reveals the identity of consecutive amino acids from C- to N-terminus:

  • Peak 1: $m/z = 175.11$. Corresponds to protonated arginine ($\text{Arg}-\text{H}^+$), establishing C-terminal Arg ($\text{R}$).

  • Peak 2: $m/z = 306.16$. Mass difference: $306.16 - 175.11 = 131.05\,\text{Da}$. Matches methionine ($\text{Met}$, $131.04\,\text{Da}$). Dipeptide = MR-COOH.

  • Peak 3: $m/z = 363.17$. Mass difference: $363.17 - 306.16 = 57.01\,\text{Da}$. Matches glycine ($\text{Gly}$, $57.02\,\text{Da}$). Tripeptide = GMR-COOH.

  • Peak 4: $m/z = 492.23$. Mass difference: $492.23 - 363.17 = 129.06\,\text{Da}$. Matches glutamate ($\text{Glu}$, $129.04\,\text{Da}$). Sequence = EGMR-COOH.

  • Peak 5: $m/z = 621.25$. Mass difference: $621.25 - 492.23 = 129.02\,\text{Da}$. Matches glutamate ($\text{Glu}$, $129.04\,\text{Da}$). Sequence = EEGMR-COOH.

  • Peak 6: $m/z = 734.33$. Mass difference: $734.33 - 621.25 = 113.08\,\text{Da}$. Matches isoleucine or leucine ($\text{Ile/Leu}$, $113.08\,\text{Da}$). Sequence = IEEGMR-COOH.

  • Peak 7: $m/z = 849.34$. Mass difference: $849.34 - 734.33 = 115.01\,\text{Da}$. Matches aspartate ($\text{Asp}$, $115.03\,\text{Da}$). Sequence = DIEEGMR-COOH.

  • Peak 8: $m/z = 950.36$. Mass difference: $950.36 - 849.34 = 101.02\,\text{Da}$. Matches threonine ($\text{Thr}$, $101.05\,\text{Da}$). Sequence = TDIEEGMR-COOH.

  • Peak 9: $m/z = 1047.43$. Mass difference: $1047.43 - 950.36 = 97.07\,\text{Da}$. Matches proline ($\text{Pro}$, $97.05\,\text{Da}$). Sequence = PTDIEEGMR-COOH.

  • Peak 10: $m/z = 1134.58$. Mass difference: $1134.58 - 1047.43 = 87.15\,\text{Da}$. Matches serine ($\text{Ser}$, $87.03\,\text{Da}$). Sequence = SPTDIEEGMR-COOH.

  • Complete deduced tryptic peptide sequence (from N- to C-terminus): V-S-P-T-D-I-E-E-G-M-R.

Amino Acid Sequences, Sequence Alignment, and Molecular Evolution

• Relative Abundance of Amino Acids in Proteins:

  • Natural occurrence frequencies across the Swiss-Prot database show distinct distribution patterns:

  • Aliphatic: Leucine ($\text{Leu}$, $9.7\%$), Alanine ($\text{Ala}$, $8.3\%$), Glycine ($\text{Gly}$, $7.1\%$), Valine ($\text{Val}$, $6.9\%$), Isoleucine ($\text{Ile}$, $6.0\%$), Proline ($\text{Pro}$, $4.7\%$).

  • Acidic: Glutamate ($\text{Glu}$, $6.8\%$), Aspartate ($\text{Asp}$, $5.5\%$).

  • Small Hydroxyl: Serine ($\text{Ser}$, $6.6\%$), Threonine ($\text{Thr}$, $5.4\%$).

  • Basic: Lysine ($\text{Lys}$, $5.9\%$), Arginine ($\text{Arg}$, $5.6\%$), Histidine ($\text{His}$, $2.3\%$).

  • Amide: Asparagine ($\text{Asn}$, $4.1\%$), Glutamine ($\text{Gln}$, $4.0\%$).

  • Aromatic: Phenylalanine ($\text{Phe}$, $3.9\%$), Tyrosine ($\text{Tyr}$, $2.9\%$), Tryptophan ($\text{Trp}$, $1.1\%$).

  • Sulfur-containing: Methionine ($\text{Met}$, $2.4\%$), Cysteine ($\text{Cys}$, $1.4\%$).

• Sequence Homology and Algorithmic Sequence Alignment:

  • Homologous Proteins: Proteins derived from a shared common evolutionary ancestor showing significant sequence conservation.

  • Aligning the catalytic nucleotide-binding site of ATP synthase between an archaeon (Sulfolobus acidocaldarius) and a bacterium (Escherichia coli) requires the algorithmic insertion of a three-residue gap into the archaeal sequence to maximize the alignment of catalytic residues.

• Substitution Scoring Matrices (BLOSUM62):

  • Alignment algorithms quantify protein sequence relationships using substitution scoring matrices such as BLOSUM62 (Blocks Substitution Matrix 62), standard in BLAST.

  • Derived from observed substitution frequencies within conserved sequence blocks sharing at least $62\%$ identity.

  • Conservative mutations between chemically similar amino acids receive positive scores (e.g., $\text{Ile} \leftrightarrow \text{Leu} = +2$, $\text{Lys} \leftrightarrow \text{Arg} = +2$, $\text{Glu} \leftrightarrow \text{Asp} = +2$), whereas nonconservative replacements receive negative penalties (e.g., $\text{Trp} \leftrightarrow \text{Asp} = -4$). Invariant, structurally unique residues carry high positive identity scores (e.g., $\text{Trp} \leftrightarrow \text{Trp} = +11$, $\text{Cys} \leftrightarrow \text{Cys} = +9$).

• Orthology and Molecular Phylogeny of Cytochrome c:

  • Orthologous Proteins: Homologs performing the same biological function in different organisms.

  • Across more than $40$ eukaryotic species, cytochrome c contains exactly $28$ strictly invariant amino acid residues: $\text{Gly}1$, $\text{Gly}6$, $\text{Phe}10$, $\text{Cys}17$, $\text{His}18$, $\text{Gly}29$, $\text{Pro}30$, $\text{Leu}32$, $\text{Gly}34$, $\text{Arg}38$, $\text{Gly}41$, $\text{Gly}45$, $\text{Tyr}48$, $\text{Asn}52$, $\text{Trp}59$, $\text{Leu}68$, $\text{Asn}70$, $\text{Pro}71$, $\text{Lys}72$, $\text{Lys}73$, $\text{Tyr}74$, $\text{Pro}76$, $\text{Thr}78$, $\text{Lys}79$, $\text{Met}80$, $\text{Phe}82$, $\text{Gly}84$, and $\text{Arg}91$.

• The number of amino acid differences between the cytochrome c sequences of any two species is proportional to their phylogenetic divergence time, functioning as a molecular evolutionary clock.

• Globin Evolution and Divergent Structural Homology:

  • Paralogous Proteins: Homologous proteins that arose through gene duplication events within a single species and diverged to acquire specialized functions.

  • An ancestral globin gene duplicated to produce modern myoglobin and an ancestral hemoglobin gene; the ancestral hemoglobin subsequently duplicated into ancestral $\alpha$-globin and $\beta$-globin genes.

  • The three-dimensional tertiary structures of horse methemoglobin $\alpha$-chain, horse methemoglobin $\beta$-chain, and sperm whale myoglobin remain almost identical despite sequence divergence.

• Structural Homology with Divergent Biological Functions:

  • Hen egg white lysozyme ($129$ residues; hydrolyzes bacterial peptidoglycan) and human milk $\alpha$-lactalbumin ($123$ residues; regulatory subunit of lactose synthase) share identical residues at $48$ sequence positions ($39\%$ identity).

  • Despite non-overlapping functions, their tertiary structures are superimposable, confirming descent from a common ancestral gene.

Chemical Synthesis of Polypeptides: Solid-Phase Peptide Synthesis (SPPS)

• Principles of Solid-Phase Synthesis:

  • Developed by R. Bruce Merrifield, solid-phase peptide synthesis (SPPS) synthesizes polypeptides in the laboratory in the $\text{C}\rightarrow\text{N}$ direction, opposite to biological ribosomal synthesis ($\text{N}\rightarrow\text{C}$).

  • The C-terminal carboxyl group of the initial amino acid is anchored covalently to an insoluble synthetic polymeric resin particle.

  • Because the growing polypeptide chain remains tethered to the resin, soluble reaction byproducts, excess coupling reagents, and unreacted monomers are washed away at each cycle by simple filtration, enabling automation for synthesizing chains exceeding $30$ residues.

• Step-by-Step SPPS Chemical Reaction Cycle:

  • Step 1: An incoming amino acid whose $\alpha$-amino group is blocked by a 9-fluorenylmethoxycarbonyl (Fmoc) group, and whose reactive side-chain moieties are blocked by acid-labile protecting groups (such as tert-butyl, $t$-butyl), is activated at its C-terminus using diisopropylcarbodiimide (DIPCDI).

  • Step 2: The activated $O$-acylisourea intermediate undergoes nucleophilic attack by the free $\alpha$-amino group of the resin-bound peptide, forming a peptide bond and releasing diisopropylurea as an insoluble byproduct.

  • Step 3: Mild base treatment (piperidine) removes the base-labile Fmoc protecting group from the newly incorporated N-terminal residue.

  • Step 4: The resin particles are rinsed, and the next Fmoc-protected, DIPCDI-activated amino acid is introduced.

  • Step 5: After complete chain assembly, treatment with concentrated acid (trifluoroacetic acid, TFA) hydrolyzes the ester linkage anchoring the C-terminus to the resin and cleaves all side-chain protecting groups, releasing the unprotected, biologically active peptide.

Nonprotein Chemical Moieties: Conjugated Proteins and Post-Translational Modifications

• Conjugated Proteins and Prosthetic Groups:

  • Conjugated Protein: A functional protein containing a non-amino acid chemical group.

  • Prosthetic Group: A permanently bound non-amino acid moiety (covalent or noncovalent) that is required for biological activity.

  • Classes of Conjugated Proteins:

  • Lipoproteins: Associated with lipid molecules (e.g., cholesterol, phospholipids, triacylglycerols) covalently or noncovalently. Examples: Blood plasma lipoprotein complexes (High-Density Lipoprotein, HDL; Low-Density Lipoprotein, LDL).

  • Nucleoproteins: Associated noncovalently with nucleic acids (RNA or DNA). Examples: Ribosomes (ribosomal ribonucleoprotein complexes), chromosomes (histone-DNA assemblies).

  • Glycoproteins: Covalently linked to carbohydrate chains (oligosaccharides). Examples: Immunoglobulins (antibodies), cell-surface receptors (e.g., LDL receptor).

  • Metalloproteins and Metal-Activated Proteins: Coordinate metal ions ($\text{Ca}^{2+}$, $\text{K}^+$, $\text{Fe}^{2+}$, $\text{Zn}^{2+}$, $\text{Co}^{2+}$) covalently or noncovalently. Examples: Metabolic enzymes, protein kinases, phosphatases.

  • Hemoproteins: Coordinate an iron-porphyrin heme ring covalently or noncovalently. Examples: Hemoglobin, myoglobin, cytochromes.

  • Flavoproteins: Contain flavin nucleotides (flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) bound covalently or noncovalently. Examples: Electron-transfer dehydrogenases.

• Prominent Post-Translational Modifications (PTMs):

  • PTMs occur after ribosomal polypeptide synthesis, modifying protein charge, conformation, localization, and activity:

  • Phosphorylation: Addition of phosphate ($-\text{PO}_3^{2-}$) to the hydroxyl groups of Ser, Thr, or Tyr residues. Examples: Regulatory enzymes, hormone receptors.

  • Acetylation: Addition of an acetyl group ($-\text{COCH}_3$) to the $\epsilon$-amino group of Lys residues. Examples: Nuclear histones, metabolic enzymes.

  • Methylation: Addition of a methyl group ($-\text{CH}_3$) to Lys or Arg residues. Examples: Histones.

  • Acylation: Covalent attachment of fatty acids (e.g., palmitic acid) to Cys side chains. Examples: G-protein-coupled receptors (GPCRs).

  • Prenylation: Covalent addition of an isoprenoid lipid group (e.g., farnesyl or geranylgeranyl) to Cys side chains. Examples: Ras p21 GTPase.

  • ADP-Ribosylation: Transfer of ADP-ribose from $\text{NAD}^+$ to His or Arg residues. Examples: Trimeric G proteins, eukaryotic elongation factor 2 (eEF-2).

  • Adenylylation (AMPylation): Transfer of an AMP moiety to the phenolic hydroxyl group of Tyr. Examples: Glutamine synthetase.

Biological Functions of Proteins and Quantitative Ligand Binding Thermodynamics

• Functional Classification of Cellular Proteins:

  • Enzymes: Catalyze biological transformations with high specificity (e.g., DNA polymerase, chymotrypsin).

  • Regulatory Proteins: Modulate cellular processes, metabolic pathways, and transcription (e.g., insulin, transcription factors).

  • Transport Proteins: Facilitate the delivery of essential substances across membranes or through circulatory fluids (e.g., hemoglobin, ion transporters).

  • Storage Proteins: Act as reservoirs of amino acids or essential metal ions (e.g., ovalbumin, ferritin, casein).

  • Contractile and Motile Proteins: Mediate cellular motion, chromosomal division, and macroscopic muscle contraction (e.g., actin, myosin, tubulin).

  • Structural Proteins: Provide mechanical stiffness, architectural integrity, and tensile strength (e.g., collagen, keratin, fibroin).

  • Scaffold and Adapter Proteins: Organize multi-protein signaling pathways into localized regulatory complexes.

  • Protective and Exploitive Proteins: Defend organisms against pathogens or facilitate predation (e.g., antibodies, blood clotting factors, venom toxins).

  • Exotic Proteins: Display specialized adaptations, such as antifreeze glycoproteins in Antarctic fish and green fluorescent protein (GFP).

• Thermodynamic Model of Protein-Ligand Binding:

  • Protein function requires specific, noncovalent binding to a target ligand ($\text{L}$):   $\text{P} + \text{L} \rightleftharpoons \text{PL}$

  • The reversible dissociation process is quantified by the dissociation constant ($K_D$):   $\text{PL} \rightleftharpoons \text{P} + \text{L}$   $K_D = \frac{[\text{P}][\text{L}]}{[\text{PL}]}$

  • Fractional Saturation ($\nu$): The fraction of total protein sites bound to ligand:   $\nu = \frac{[\text{PL}]}{[\text{P}] + [\text{PL}]}$

• Substituting $[\text{P}] = \frac{K_D [\text{PL}]}{[\text{L}]}$ yields the hyperbolic binding isotherm equation: $\nu = \frac{[\text{PL}]}{\frac{K_D [\text{PL}]}{[\text{L}]} + [\text{PL}]} = \frac{[\text{L}]}{K_D + [\text{L}]}$

• Half-Saturation Mechanics:

  • When fractional saturation is half-maximal ($\nu = 0.5$):     $0.5 = \frac{[\text{L}]}{K_D + [\text{L}]} \implies K_D + [\text{L}] = 2[\text{L}] \implies [\text{L}] = K_D$

• The dissociation constant ($K_D$) is equal to the concentration of free ligand at which half of the total protein binding sites are occupied.

• Lower values of $K_D$ reflect higher binding affinity.

Proteomics and High-Throughput LC-MS/MS Analysis

• Genome versus Proteome:

  • The genome encompasses the static blueprint of all genetic potential encoded in an organism's DNA.

  • The proteome is the dynamic, functional complement of all proteins expressed in a specific cell type, under defined physiological parameters, at a specific moment in time.

  • Due to alternative RNA splicing, RNA editing, and diverse post-translational modifications, a genome containing approximately $20,000$ genes can generate a proteome containing hundreds of thousands of distinct functional proteoforms.

• High-Throughput Shotgun Proteomics Workflow (MudPIT / LC-MS/MS):

  • Step 1: Proteins extracted from a cellular lysate are digested with endoproteases (e.g., trypsin) into a complex mixture of peptides.

  • Step 2: Peptides are loaded offline onto a biphasic microcapillary column containing Strong Cation Exchange (SCX) and Reversed-Phase (RP) matrices (Multidimensional Protein Identification Technology, MudPIT).

  • Step 3: Peptides are resolved sequentially by charge and hydrophobicity and injected online into an electrospray ionization tandem mass spectrometer (LC-MS/MS).

  • Step 4: Individual peptides are selected in $\text{MS-1}$, fragmented in the collision cell via collision-induced dissociation, and product ions are resolved in $\text{MS-2}$.

  • Step 5: High-throughput computational algorithms match experimental fragmentation spectra against theoretical spectra generated from translated genomic databases, identifying and quantifying the proteins present within the original cellular extract.