Video Notes: Water, Weak Interactions, Ionization, Buffers; Amino Acids, Peptides, and Proteins (Vocabulary Flashcards)
2.1 Weak Interactions in Aqueous Systems
Water is the most abundant substance in living systems; ~70%+ of weight in many organisms.
Water shaped evolution of biochemistry; life began in aqueous environments.
Key ideas emphasized:
Water’s solvent properties and hydrogen bonding govern biomolecule structure, self-assembly, and interactions.
Ionization of water and of weak acids/bases in water determine pH, buffering, and titration behavior.
Noncovalent interactions (hydrogen bonds, ionic interactions, hydrophobic effect, van der Waals) are individually weak but collectively drive biomolecular architecture.
Water molecule and ionization products (H+ and OH−) profoundly influence structure and function of proteins, nucleic acids, lipids.
Hydrogen bonding in water: water–water interactions create cohesion; hydrogen bonds can form with solutes; water can donate/accept hydrogen bonds.
Water’s properties arising from intermolecular attractions:
Higher melting point, boiling point, and heat of vaporization than many solvents; due to extensive hydrogen bonding networks.
Bond strength: hydrogen bonds have typical bond energy ~23 kJ/mol; covalent O–H ≈ 470 kJ/mol; C–C ≈ 350 kJ/mol; hydrogen bond ~10% covalent in character.
Water structure and bonds:
Water geometry: H–O–H bond angle ≈ 104.5° (slightly less than tetrahedral 109.5° due to lone pair repulsion).
Oxygen is more electronegative; shared electrons dwell more around O, creating dipoles.
Each water molecule can form up to ~4 hydrogen bonds; in liquid water, average ~3.4 hydrogen bonds per molecule; in ice, a full tetrahedral network (4 H-bonds) forms a regular lattice.
Lifetime of a hydrogen bond in liquid water ~1–20 ps; rapid breaking/forming leads to “flickering clusters.”
Water as solvent for polar solutes; hydrophilic vs hydrophobic solubility:
Polar biomolecules dissolve readily due to favorable water–solute interactions.
Nonpolar molecules disrupt water’s network and cluster together (hydrophobic effect); poorly soluble in water.
Amphipathic molecules have polar/charged regions and nonpolar regions; their aqueous behavior includes micelle formation and, in membranes, bilayer formation driven by the hydrophobic effect.
Water forms hydrogen bonds with polar solutes (Fig. 2-3, 2-4 reference):
Alcohols, aldehydes, ketones, and N–H containing compounds form hydrogen bonds with water; C–H bonds are poor hydrogen-bond donors.
Solubility and dielectric screening:
Water dissolves salts (e.g., NaCl) by hydrating ions; ions leave crystal lattice and water stabilizes, increasing entropy.
Dielectric constant of water: ε ≈ 78.5 at 25 °C; benzene ε ≈ 4.6.
Ionic interactions depend on charges, distance r, and solvent dielectric: F = (Q1 Q2) / (ε r^2).
In water, screening of charge is strong; in nonpolar environments, ionic interactions are stronger due to less screening.
Thermodynamics of dissolution and hydrophobic solvation:
Dissolution of polar/charged solutes generally has small positive ΔH but large positive ΔS, making ΔG negative overall.
Hydrophobic solutes disrupt water’s hydrogen-bonding network, causing ordering of surrounding water (clathrate-like shell) and an entropic penalty; dissolution is energetically unfavorable unless hydrophobic surfaces cluster to reduce the ordered water shell (hydrophobic effect).
Nonpolar gases are poorly soluble in water due to both their nonpolarity and entropy decrease when entering solution; some gases (NH3, NO, H2S) are polar and can dissolve readily.
Hydration and micelles:
Amphipathic molecules form micelles in water; hydrophobic tails hide inside, polar heads face outwards, increasing entropy by releasing ordered water.
Bound water:
Water molecules can bind tightly to biomolecules (e.g., hemoglobin); bound water is not osmotically active and can be essential to function (e.g., proton hopping in cytochrome f).
van der Waals interactions are weak but contribute to macromolecular packing and stabilization in biomolecules.
Osmosis and colligative properties:
Solutes change solvent properties (vapor pressure, boiling point, freezing point, osmotic pressure).
Colligative properties depend on solute particle number, not identity: Π = i c R T, or more generally Π = RT (i1 c1 + i2 c2 + … + incn).
van’t Hoff factor i accounts for dissociation (e.g., NaCl i = 2; nonelectrolytes i = 1).
Osmosis: water moves to equalize solute concentration across semipermeable membranes; osmotic pressure Π is the force needed to stop water flow.
Isotonic solutions have osmolarity close to cytosol; hypertonic vs hypotonic describe water movement directions.
Worked Example (SUMMARY 2.1, Worked EX 2-1):
Determine osmotic strength of lysosomes given solutes; use Π = RT ∑ i c; equate to a sucrose solution to find csucrose.
Example calculation yields csucrose ≈ 0.26 M and mass ~88.9 g/L; rounded to correct significant figures.
Summary points:
Water’s polarity and hydrogen-bonding drive solvation; weak interactions are numerous and collectively critical for folding and assembly of macromolecules.
Water’s dielectric properties screen charges; osmotic pressure is a key cellular driver; hydrophobic effect underpins membrane formation and protein folding.
2.2 Ionization of Water, Weak Acids, and Weak Bases
Reversible ionization of water: H2O ⇌ H+ + OH−; in solution, hydroxonium H3O+ is the hydrated form of H+.
Ion mobility: hydronium and hydroxide have high ionic mobility due to proton hopping (Grotthuss mechanism); relative to Na+ or Cl−, H+ moves unusually quickly.
Equilibrium constants for reversible reactions: Keq in the generic form A + B ⇌ C + D is Keq = ([C][D])/([A][B]); in nonideal solutions, activities are used, but molar concentrations are commonly used in teaching.
Ion product of water at 25 °C:
Kw = [H+][OH−] = 1.0 × 10^−14 M^2.
This arises from Kw = (55.5 M)(Keq) with [H2O] ≈ 55.5 M; Keq ≈ 1.8 × 10^−16 M at 25 °C.
pH and pOH concepts:
pH = −log[H+]; pOH = −log[OH−]; pH + pOH = 14 at 25 °C.
The pH of pure water at 25 °C is 7.0 (neutral).
Worked Examples (Ionization concepts):
Given [OH−] = 0.1 M (from strong base), [H+] = Kw / [OH−] = 1.0 × 10^−14 / 0.1 = 1.0 × 10^−13 M.
Given [H+] = 1.3 × 10^−4 M, [OH−] = Kw / [H+] ≈ 7.7 × 10^−11 M.
The pH scale values and buffering implications: buffers resist pH changes by shifting equilibria; pKa of conjugate pairs and Henderson–Hasselbalch are central to buffering behavior.
pH scale details and common fluids (Table 2-5): pH ranges and corresponding [H+] and [OH−] values; pH + pOH = 14.
pH measurement: glass electrode pH meters are used to measure pH; pH affects structure and activity of biomolecules; clinical contexts include acidosis and alkalosis.
The Henderson–Hasselbalch equation for weak acids and bases: Ka = ([H+][A−])/[HA], leading to pH = pKa + log([A−]/[HA]).
Titration curves for weak acids and bases reveal the pKa as the pH at the midpoint (where [HA] = [A−]). Example: acetic acid (pKa ≈ 4.76) with NaOH titration; the midpoint pH equals pKa.
Physiological buffer systems and pH regulation:
Phosphate system: H2PO4− ⇌ H+ + HPO4^2−; pKa ≈ 6.86; buffering between pH ≈ 5.9–7.9.
Ammonium system: NH4+ ⇌ NH3 + H+; pKa ≈ 9.25; buffering between pH ≈ 8.3–10.3.
Blood bicarbonate buffer system:
H2CO3 ⇌ H+ + HCO3−; CO2 dissolution/carbondioxide hydration links to pH via CO2 partial pressure (pCO2).
In blood, CO2/gas phase and dissolved CO2 equilibria provide a large reservoir of H2CO3; pH ~ 7.4 is maintained by respiratory (CO2) control and bicarbonate buffering.
Apparent pKa for CO2 in blood (pKcombined ≈ 6.1; Kh and Ka combined):
CO2(aq) in equilibrium with H2CO3; apparent pK ≈ 6.1 at 37 °C; blood pH ≈ 7.4 when [HCO3−] ≈ [H2CO3] × 20.
Buffering power and Henderson–Hasselbalch implications: buffers are most effective around their pKa; phosphate buffers around pH 6–7; bicarbonate system buffers near physiological pH.
Boxed Medicine and historical notes summarize clinical implications of pH, buffering, and respiratory regulation (e.g., Haldane and Davies self-experiments; diurnal and disease-related pH shifts).
2.3 Buffering against pH Changes in Biological Systems
Biological systems are highly pH-sensitive; many enzymes and biomolecules require near-neutral pH for function.
Buffers are mixtures of a weak acid and its conjugate base; they resist pH changes upon addition of acid or base.
Example: Acetate buffer (HAc/Ac−) at midpoint pH ≈ pKa; buffering capacity is maximal at the midpoint where [HAc] ≈ [Ac−].
Buffer action is explained by two concurrent equilibria (Kw and Ka): adding H+ or OH− shifts the ratio [HA]/[A−] to restore equilibrium.
Henderson–Hasselbalch equation for buffers: pH = pKa + log([A−]/[HA]).
Specific biological buffers:
Phosphate buffer system: H2PO4− ⇌ H+ + HPO4^2−; pKa ≈ 6.86; effective around pH 5.9–7.9.
NH4+/NH3 buffer: pKa ≈ 9.25; effective around pH 8.3–10.3.
Bicarbonate/CO2 system: pKcombined ≈ 6.1 (37 °C); balance between HCO3− and CO2(aq) sets blood pH near 7.4.
Worked Example (Phosphate Buffers): For NaH2PO4/Na2HPO4 mixtures, pH ≈ pKa + log([HPO4^2−]/[H2PO4−]); example yields pH ≈ 7.0 for 0.042 M NaH2PO4 and 0.058 M Na2HPO4.
Practical questions illustrate buffer behavior in more complex contexts (e.g., adding NaOH to buffer, pH of buffered vs. unbuffered water).
The pH of blood and physiological buffering depend on CO2 levels and bicarbonate concentration; CO2 is rapidly exchanged in lungs to regulate buffering capacity.
Glycine, histidine, phosphate, and bicarbonate buffers contribute to intracellular and extracellular buffering; histidine’s imidazole side chain (pKa ≈ 6.0) provides significant buffering near neutral pH.
Summary (2.2–2.3):
Water’s ionization and buffering principles are central to understanding pH, buffer systems, and biological regulation.
Henderson–Hasselbalch provides a practical framework for predicting pH in buffers and biological systems.
Biological buffering relies on multiple systems (phosphate, bicarbonate, protein side chains, intracellular and extracellular fluids) to maintain homeostasis around pH ~ 7.0–7.4.
3.1 Amino Acids
Amino acids: 20 standard α-amino acids; all are α-amino acids with carboxyl and amino groups attached to the α-carbon; side chain R varies among amino acids.
α-Carbon: chiral center (except glycine, which has two hydrogens on the α-carbon); results in two enantiomers (L and D).
Absolute configuration and nomenclature:
Fischer convention relates to glyceraldehyde; L vs D depends on the configuration relative to L-glyceraldehyde (not on optical rotation).
In biology, proteins contain almost exclusively L-amino acids (D-amino acids are rare, e.g., in some peptides produced posttranslationally).
Stereochemical conventions: L- vs D- (α-amino group positions), RS system for absolute configuration; glycine is not chiral.
Amino acid properties and classification (Table 3-1 overview):
pK1 (−COOH) and pK2 (−NH3+) values vary by residue; pKR denotes pKa of ionizable R group; pI is isoelectric point (average of relevant pKa values for amino acids with ionizable R groups).
Five major classes by R-group polarity: nonpolar, aromatic, polar uncharged, positively charged, negatively charged.
Arguably most important features for structure/function: hydrophobicity, charge distribution, capacity to form hydrogen bonds, and acid/base properties.
Aromatic residues and UV absorption:
Tyr, Trp, and Phe absorb UV light; Trp and Tyr contribute strongly around 280 nm; used in protein quantification by UV absorbance.
Special residues and posttranslationally modified amino acids:
Cysteine can form disulfide bonds (Cys–Cys) and can be modified to form cystine; cysteine is involved in disulfide cross-links stabilizing protein structure.
Selenocysteine and pyrrolysine are special cases integrated via unique cellular machinery.
Other uncommon residues: hydroxyproline, γ-carboxyglutamate, desmosine; posttranslational modifications regulate activity.
Zwitterionic nature and buffering roles:
In aqueous solution at neutral pH, amino acids exist as zwitterions: +NH3–CH(R)–COO−; act as buffers when R-group pKa or terminal groups are near physiological pH.
Charge states and pI:
The net charge of an amino acid depends on pH relative to pKa values; pI is the pH at which net charge is zero; for glycine pI ≈ (pK1 + pK2)/2 ≈ 5.97.
Two-Dimensional content on amino acids (Table 3-1 values):
Examples: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F), Tyr (Y), Trp (W), Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q), Lys (K), His (H), Arg (R), Asp (D), Glu (E).
Practical implications:
pKa perturbations arise from local environment (electronegativity of neighboring groups, intramolecular interactions) affecting ionization vs. standard model compounds.
Histidine’s side chain with pKa ≈ 6.0 provides buffering near neutral pH; many enzymes exploit this in catalysis.
3.2 Peptides and Proteins
Peptides and polypeptides:
Amino acids join via peptide bonds (amide linkages) formed by condensation (water removal); residues are what remain after dehydration.
A dipeptide has two amino acids linked by a peptide bond; oligopeptides are short chains; polypeptides are long chains and constitute proteins.
N-terminus (amino-terminal) has free α-amino group; C-terminus (carboxyl-terminal) has free α-carboxyl group.
Peptide bond properties:
Peptide bond is planar with partial double-bond character; rotation around the peptide bond is limited (ω ≈ 180° for trans; cis for rare cases, especially with Pro).
The backbone dihedral angles define conformation: ϕ (phi) about N–Cα; ψ (psi) about Cα–C; ω is the peptide bond; ω is almost always trans (~99.6%); cis occasionally occurs at proline-containing bonds.
Polypeptide architecture and nomenclature:
The sequence is read from N-terminus to C-terminus; residues are denoted by one-letter or three-letter codes (Table 3-1 conventions).
The term residue reflects loss of water during peptide bond formation (H2O removed).
Ionizable residues in peptides:
Peptides retain ionizable side chains which contribute to overall acid-base behavior and titration curves; terminal groups remain ionizable unless involved in a peptide bond.
Protein size and composition:
Proteins can range from small peptides to giant assemblies; average protein residual weight and composition differ; glycine average mass ~128 Da for residues; water loss reduces mass per residue to ≈ 128 − 18 = 110 Da.
Post-translationally modified residues and nonstandard amino acids:
Many proteins incorporate nonstandard residues or prosthetic groups (e.g., lipoproteins, glycoproteins, metalloproteins, etc.).
The A and B chains of insulin (Sanger’s classic work) illustrate peptide sequencing and chain architecture; proteolytic fragmentation and degradation enable sequencing and the mapping of disulfide linkages.
Practical notes:
Peptides can be chemically synthesized (Merrifield solid-phase method) and then ligated to form longer proteins; synthesis efficiency affects yield of full-length products.
Peptide sequencing and protein sequencing rely on Edman degradation, protease digestion, and modern MS techniques (peptide fragmentation patterns, b- and y-ions).
3.3 Working with Proteins
Purification principles:
Proteins are purified by exploiting differences in size, charge, solubility, binding properties, and affinity.
Common methods: salting out (ammonium sulfate precipitation), dialysis, and various chromatography techniques (Table 3-5, Fig. 3-16 series).
Chromatography types:
Ion-exchange chromatography: separates by net charge at a given pH via cation- or anion-exchange resins; elution is achieved by changing pH or salt gradient.
Size-exclusion (gel-filtration) chromatography: separates by hydrodynamic volume/size; larger proteins elute earlier because they do not enter small pores.
Affinity chromatography: binds proteins by specific interactions with a ligand; elution occurs with a free ligand or high salt.
High-performance liquid chromatography (HPLC): used to improve resolution and speed; often used in combination with other chromatography steps.
Protein analysis and monitoring purification:
Electrophoresis: SDS-PAGE separates by molecular weight; denatures proteins with SDS to normalize charge-to-mass ratio; Coomassie blue staining visualizes bands; 2D electrophoresis combines isoelectric focusing with SDS-PAGE for higher resolution.
Isoelectric focusing (IEF): separates by isoelectric point (pI) on a pH gradient; can be combined with SDS-PAGE for 2D separation.
Multistep purification tables document fractions, total protein, activity, specific activity, and purification factor; yield is tracked as a percent of starting activity.
Activity assays and quantification:
Enzyme activity is defined as amount of substrate converted per unit time; specific activity is activity per mg protein; purification increases specific activity as contaminants are removed.
For non-enzymes, other functional assays or binding measurements can quantify protein content.
Analytical methods and modern approaches:
Mass spectrometry (MALDI, ESI, tandem MS) identifies proteins and sequences, measures exact masses, and provides proteome coverage.
2D coupling of MS with LC (LC-MS/MS) enables complex mixture analysis and quantitation of protein abundance.
Protein purification strategy:
Purification is often sequential, leveraging different properties (size, charge, affinity); a typical workflow uses inexpensive steps first (salting out) and saves expensive methods for later stages.
3.4 The Structure of Proteins: Primary Structure
Primary structure refers to the linear sequence of amino acids in a protein and the covalent linkages (peptide bonds and disulfide bonds) that connect them.
Key historical and conceptual points:
The sequence determines three-dimensional structure and function; Sanger’s insulin sequencing demonstrated the information content of a protein sequence.
The central dogma linking genotype to phenotype relies on the sequence-to-structure-to-function relationship.
Stereochemistry and amino acid form in proteins:
Proteins predominantly contain L-amino acids; D-amino acids are rare and usually not part of proteins.
The amino acid sequence encodes structural motifs and functional domains; evolution preserves important residues in conserved regions and may tolerate variation elsewhere.
The concept of motifs and domains:
Motifs (folds) are recurring structural patterns composed of one or more secondary-structure elements;
Domains are independently folding units that can be modularly rearranged; proteins often contain multiple domains with distinct functions.
Post-translationally modified amino acids:
Proteins can contain uncommon residues (e.g., hydroxyproline, γ-carboxyglutamate), and some residues may be formed by posttranslational modification (phosphorylation, methylation, acetylation, etc.).
The role of sequence in function and evolution:
Protein sequences enable evolutionary tracing, detection of homologous proteins, and inference of functional relationships.
The concept of orthologs (across species) and paralogs (within a species) helps map protein evolution.
Practical issues:
Determination of sequence can be achieved through direct sequencing (Edman degradation for early work) or modern mass spectrometry; sequencing underpins database-driven function prediction.
The structure–function–evolution relationship forms the basis for understanding protein families, fold conservation, and domain architectures.
4.1 Overview of Protein Structure
Proteins possess hierarchical structure: primary, secondary, tertiary, and quaternary levels.
Four major classes of proteins by structure: fibrous, globular, membrane, and intrinsically disordered.
Key ideas:
Fibrous proteins (e.g., α-keratin, collagen, silk fibroin) have simple repeating elements and often structural roles; they are typically insoluble due to hydrophobic surfaces and cross-linking.
Globular proteins are compact, diverse in function (enzymes, transport, regulation); folding is driven by weak interactions and hydrophobic collapse, yielding a hydrophobic core.
Membrane proteins are embedded in lipid bilayers; often contain hydrophobic transmembrane segments; discussed further in Chapter 11.
Intrinsically disordered proteins lack fixed 3D structure yet have essential regulatory and interaction roles; their sequences tend to be charged and flexible.
Stabilizing forces in folded proteins:
Hydrophobic effect drives burial of nonpolar residues in a hydrophobic core, increasing solvent entropy.
Hydrogen bonds and ionic interactions (salt bridges) stabilize specific orientations and interactions within and between motifs.
van der Waals interactions contribute collectively in densely packed cores.
Disulfide bonds (covalent) can stabilize structures; more common in extracellular proteins, and in some thermophiles.
The concept of proteostasis:
Proper protein folding requires chaperones (e.g., Hsp70, chaperonins GroEL/GroES) and quality-control pathways to prevent misfolding and aggregation.
Misfolded proteins can lead to disease (amyloidoses, prion diseases, cystic fibrosis, etc.).
Protein structure determination methods (overview):
X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, cryo-electron microscopy (cryo-EM) are principal structural biology tools.
Structural data are archived in the Protein Data Bank (PDB); tools such as SCOP2 classify motifs and folds; consensus sequences and sequence logos summarize conserved patterns.
Fold and motif concepts:
Motifs/folds are recurring 3D arrangements (e.g., α/β barrel) that recur across different proteins and reflect conserved functional strategies.
Domains are autonomous folding units; many proteins contain multiple domains that contribute to function and regulation.
Protein modeling and modern advances:
Computational biology (molecular modeling, dynamics, and crowdsourcing platforms like Foldit) contributes to protein design and understanding folding landscapes.
4.2 Protein Secondary Structure
Secondary structure comprises regular, repeating backbone conformations: α-helix, β-conformation (β-sheet), and β-turns.
α-Helix:
Right-handed helix is the common form in proteins; repeating unit 3.6 residues per turn; rise per residue ≈ 1.5 Å; overall rise per turn ≈ 5.4 Å.
Intramolecular hydrogen bonding pattern: every peptide bond participates in H-bonds with the 4th residue ahead, stabilizing the helix.
End-capping: n- and c-termini contribute to helix dipole; negatively charged residues near the N-terminus and positively charged residues near the C-terminus can stabilize/destabilize via interactions with the helix dipole.
Proline disrupts helices due to rigid ring and lack of amide NH for hydrogen bonding; glycine is flexible and often disfavors helices.
β-conformation (β-sheet):
Extended zigzag arrangement; β-strands align side-by-side to form β-sheets; strands can be parallel or antiparallel.
Interstrand hydrogen bonds stabilize sheets; R groups alternate on opposite sides of the sheet, giving a pleated appearance.
Parallel versus antiparallel: antiparallel sheets have more linear H-bonds and generally greater stability in terms of H-bond geometry; parallel sheets have a different H-bond pattern and longer strand spacing.
Turns and loops:
Turns connect secondary structural elements; β-turns commonly connect antiparallel β-sheets and often include Gly or Pro; cis/trans isomerism around peptide bonds (Proline can adopt cis) influences turn geometry.
Ramachandran plots:
Visualize allowed ϕ (phi) and ψ (psi) dihedral angles for backbone; most data cluster around α-helix and β-sheet regions; glycine occupies broader conformational space due to small side chain.
Circular dichroism (CD) spectroscopy:
Measures differential absorption of left- vs right-circularly polarized light in the far-UV; characteristic spectra distinguish α-helix, β-sheet, and random coil; used to estimate fraction of secondary structure and to monitor folding/denaturation.
4.3 Protein Tertiary and Quaternary Structures
Tertiary structure: the overall 3D arrangement of a single polypeptide chain; composed of multiple secondary structure elements folded into a stable core.
Quaternary structure: arrangement of multiple polypeptide chains (subunits/protomers) into a multisubunit complex.
Stabilizing features in 3D structure:
Hydrophobic core formation buries nonpolar residues away from water.
Hydrogen bonds and ionic interactions help organize and stabilize tertiary structure; salt bridges can be particularly stabilizing in low-dielectric environments (e.g., protein interior).
Disulfide bonds provide covalent cross-links that can lock conformations; more common in extracellular and thermophilic proteins.
Major structural classes:
Fibrous proteins: elongated, repetitive secondary structure; often insoluble; examples include α-keratin, collagen, silk fibroin.
Globular proteins: compact, diverse folds; many soluble enzymes and structural proteins fall into this class.
Membrane proteins: span lipid bilayers; contain hydrophobic transmembrane segments; significant for transport and signaling.
Intrinsically disordered proteins: lack fixed structure; often function as signaling hubs or regulators and can gain structure upon binding partners.
Structural motifs and domains:
Motifs/ folds are recurrent combinations of secondary structure elements with specific topology.
Domains are independently folding units; proteins often consist of multiple domains with distinct roles.
Structure–function–evolution connections:
Structural motifs are conserved across protein families; function can be inferred from conserved folds.
Protein families and superfamilies (SCOP2, etc.) help map evolutionary relationships; homology inferred from sequence and/or structural similarity.
Experimental and computational structure elucidation:
X-ray crystallography, NMR, cryo-EM, and computational modeling provide structural information.
The Protein Data Bank (PDB) stores 3D structures; PDB IDs allow retrieval of structures for study.
Ramachandran analysis, topology diagrams, and motif databases help interpret and compare structures.
4.4 Protein Denaturation and Folding
Proteostasis: the cellular balance of protein synthesis, folding, refolding, and degradation; misfolded proteins are cleared or rescued.
Protein folding is highly cooperative and fast; Levinthal’s paradox suggests folding cannot be a random search; instead, folding is hierarchical:
Local secondary structures form first, guided by dihedral angle propensities and sequence constraints.
Long-range interactions subsequently assemble into stable tertiary and quaternary structures.
Hydrophobic collapse and formation of a protein core occur early in folding; specific hydrogen-bonding networks and salt bridges refine the structure.
Folding pathways and energy landscapes:
Concept of folding funnels; the native state lies at the bottom of the funnel; intermediates may exist as local energy minima.
Some proteins fold via multiple pathways with varying intermediates; others fold with a simpler, smoother funnel.
Assisted folding and chaperones:
Hsp70 family (e.g., Hsp70, Hsp40) assists nascent peptides; chaperonins (GroEL/GroES in bacteria; Hsp60 in eukaryotes) provide protected environments for folding.
Peptidyl isomerases (PPI) catalyze cis–trans isomerization of Pro peptide bonds; PDI (protein disulfide isomerase) reshuffles disulfide bonds.
Misfolding and disease:
Misfolded proteins can aggregate into amorphous aggregates or ordered amyloid fibrils; associated with diseases like Alzheimer's, Parkinson's, Huntington’s, prion diseases, and CFTR misfolding.
Renaturation and classic experiments:
Anfinsen’s experiments with ribonuclease showed that a protein’s amino acid sequence contains information necessary to fold into its native structure; denaturation with urea/reducing agent can be reversed to restore activity.
Denaturation methods and effects:
Heat, extremes of pH, organic solvents, urea/guanidinium salts, detergents can denature proteins by perturbing hydrophobic core, disrupting hydrogen bonds, or altering charge.
Denatured proteins often precipitate; some aggregates are disordered, others form ordered structures.
Key takeaways on stability and folding:
The native state is marginally stable; small changes can alter function.
Hydrophobic effect, hydrogen bonds, and ionic interactions are central to stability; disulfide bonds contribute covalent stabilization.
Folding is a balance of enthalpic and entropic contributions; chaperones and cofactors often shape and stabilize folding pathways.
4.5 Determination of Protein and Biomolecular Structures
Structural biology integrates experimental and computational methods:
X-ray crystallography: provides high-resolution 3D structures from diffraction patterns; requires crystals; electron density maps derived by Fourier transforms model into molecular structure.
NMR spectroscopy: yields information about atoms in solution; NOE/TOCSY data provide distance constraints; useful for dynamic and flexible regions; often used for smaller proteins or domains.
Cryo-electron microscopy (cryo-EM): visualizes large complexes without crystallization; single-particle analysis yields 3D reconstructions from 2D images; increasingly high resolution.
Box 4-3: Protein Data Bank (PDB) as a central resource; PDB IDs identify structures; PDB data include coordinates, experimental details, and validation.
Computational methods and modeling:
Molecular dynamics simulations, fold prediction, and in silico design complement experimental methods.
Crowdsourced protein folding via Foldit and other platforms (e.g., Rosetta@home) illustrate the interface of computation, protein design, and experimental validation.
Emerging technologies and future directions:
Integration of multi-method approaches (X-ray, NMR, cryo-EM) for comprehensive structural understanding; continuing expansion of the PDB and improved algorithms for structure determination and validation.
Boxed concepts and data highlights (cross-cutting)
Key constants and formulas to remember:
Water ion product: K_w = [H^+][OH^-] = 1.0 imes10^{-14} ext{ M}^2 ext{ (25 °C)}
Henderson–Hasselbalch (acid): pH = pK_a + ext{log} rac{[A^-]}{[HA]}
pH scale: pH = - ext{log}{10}[H^+], pOH = - ext{log}{10}[OH^-], ext{ and } pH + pOH = 14
Osmotic pressure (ideal, van’t Hoff): oxed{oxed{
abla ext{Osmotic Pressure } \ \ \ \ \ \\Pi = i c R T}} and the multi-species version: oxed{oxed{\ \ \ \ \ \ \\Pi = RTigl(i1 c1 + i2 c2 +
ull + in cnigr)}}Dielectric screening of ionic interactions in water: F= rac{Q1 Q2}{
abla ext{?}} ext{(explicit form } F= rac{Q1 Q2}{ frac{1}{ ext{dielectric}} r^2} ext{; use } rac{Q1 Q2}{
^2
abla} ext{ with }
abla= rac{1}{ ext{ε}})Hydrophobic effect drives clustering and micelle/bilayer formation to maximize entropy of surrounding water.
Conceptual takeaways:
Weak interactions are numerous and collectively stabilize macromolecular structure and enzyme–substrate binding.
Water’s properties (polarity, hydrogen bonding, dielectric constant) shape solubility, folding, and assembly of biomolecules.
Buffers enable biological systems to maintain proper pH; major physiological buffers include phosphate and bicarbonate systems; blood pH remains around 7.4 due to integrated buffering and respiratory control.
Protein structure is analyzed through a hierarchy: primary (sequence), secondary (α-helices, β-sheets, turns), tertiary (3D fold), and quaternary (assembly of subunits).
The sequence–structure–function paradigm is foundational to biochemistry and molecular biology; evolution shapes protein families and domains.
Examples and historical notes:
Sanger’s insulin sequencing demonstrated how amino acid sequences encode protein structure and function; foundational to early protein chemistry.
Boxed medical and historical notes illustrate how pH and buffering have direct clinical relevance (acidosis, alkalosis, bicarbonate therapy).
Interfaces with modern biology: mass spectrometry (MS/MS) for sequencing, proteomics, and structure-based drug design; Foldit and Rosetta illustrate crowdsourced protein design.
Equations and constants to memorize:
Water ionization and Kw: K_w=[H^+][OH^-]=1.0 imes10^{-14} ext{ M}^2
Henderson–Hasselbalch (buffers): ext{pH}= ext{p}K_a + ext{log} rac{[A^-]}{[HA]}
pH and pKa relation: ext{p}Ka=- ext{log}{10}K_a
Buffering regions around pKa: region roughly between 10% and 90% with maximal buffering at exactly pH = pKa.
Isoelectric point for glycine: pI= rac{pK1+pK2}{2}=5.97 (example)
Glucose and glycogen osmotic considerations: mass vs osmolar contribution; polysaccharides have smaller osmotic impact than equivalent mass of monomers.
Connections to broader topics:
Ionization and buffering connect chemistry to physiology (blood pH regulation, respiration, renal function).
Water’s properties underpin biophysics of protein folding, binding, and membrane formation.
Structure determination techniques enable drug design, understanding disease mechanisms, and elucidating evolutionary histories.
Practical implications and ethical/philosophical context:
Modulating pH in biological systems must consider buffering capacity and potential toxicities (e.g., bicarbonate therapy in acidosis).
Misfolding diseases highlight the importance of proteostasis and quality control in health and aging.
Computational design and designer proteins raise ethical considerations about bioengineering and biosafety, balanced by powerful therapeutic and industrial potentials.
LaTeX quick-reference (recap):
Hydrogen ion equilibrium: K_w=[H^+][OH^-]
Henderson–Hasselbalch: pH=pK_a+ ext{log} rac{[A^-]}{[HA]}
pH definition: pH=- ext{log}_{10}[H^+]
Osmotic pressure: oxed{
\, oxed{\Pi = RT \, (i1 c1 + i2 c2 + \, \cdots + in cn)}}
Dielectric screening: F= rac{Q1 Q2}{\varepsilon r^2}
Hydrophobic effect qualitatively described as entropy-driven aggregation of nonpolar groups to minimize ordered water shells.