Unit 1: Introduction to biophysics

Biophysics applies the concepts and quantitative tools of physics—energy, force, thermodynamics, electrostatics and spectroscopy—to biological molecules and processes. It emerged as a distinct discipline in the mid-20th century (crystallography of proteins and DNA, 1950s onward) and treats the cell as a system governed by physical law rather than by vitalist principles.

• Central premise: biological function follows from molecular structure, and structure is fixed by the balance of physical forces.
• Governing energetics: processes proceed toward lower Gibbs free energy, ΔG = ΔH − TΔS, where ΔH is enthalpy, T is absolute temperature (K) and ΔS is entropy.
• Scale of interest: interactions in the range of 1–100 kJ/mol, weak enough to be broken by thermal energy at 37 °C (RT ≈ 2.5 kJ/mol).
• Aqueous context: water (dielectric constant ε ≈ 80) dominates every interaction by screening charges and driving the hydrophobic effect.
• Directionality of information: the sequence of monomers encodes biological information, transmitted DNA → RNA → protein.

II. Biomolecules

The polymeric building blocks of the cell.

Biomolecules are the carbon-based macromolecules and their monomers from which living matter is assembled.

A. Definition and the four major classes

• Proteins: polymers of 20 amino acids joined by peptide bonds; fold into defined 3-D shapes to act as enzymes, receptors and structural elements.
• Nucleic acids: polymers of nucleotides (DNA, RNA) storing and transmitting genetic information.
• Carbohydrates: polyhydroxy aldehydes/ketones (e.g. glucose, C₆H₁₂O₆) serving as fuel and structural material.
• Lipids: hydrophobic molecules (fatty acids, phospholipids) forming membranes and energy stores.

B. Monomers and the condensation principle

• Building principle: monomers link by dehydration (loss of H₂O) and break by hydrolysis (addition of H₂O).
• Amino acid backbone: central α-carbon bearing an –NH₃⁺, –COO⁻, an H, and a variable side chain R.
• Nucleotide: phosphate + pentose sugar (ribose or deoxyribose) + nitrogenous base.

C. Levels of structural organization

• Primary: covalent sequence of monomers (e.g. Met-Gly-Ser…).
• Secondary: local hydrogen-bonded motifs (α-helix, β-sheet, DNA double helix).
• Tertiary: the full 3-D fold of a single chain.
• Quaternary: assembly of multiple chains (e.g. haemoglobin = 4 subunits).

III. Chemical Bonds in Biochemistry

The interaction hierarchy that fixes and reshapes biomolecular structure.

Biological structure is maintained by a spectrum of bonds ranging from strong covalent linkages to numerous weak non-covalent interactions.

A. Covalent bonds — the strong scaffold

• Peptide and phosphodiester bonds: covalent, 200–400 kJ/mol, define primary structure and are stable to thermal motion.
• Disulfide bridge: –S–S– between two cysteine residues, covalently cross-links folded proteins.

B. Non-covalent interactions — the tunable forces

These weak, reversible interactions collectively determine folding and recognition.

• Ionic (electrostatic) bonds: attraction between charged groups, energy E = q₁q₂ / (4πε₀ε r), where q are charges, r the separation, ε the dielectric constant; strongly weakened in water.
• Hydrogen bonds: a shared H between an electronegative donor and acceptor (N–H···O), ~10–30 kJ/mol; stabilize helices and base pairs.
• Van der Waals forces: transient dipole attraction (~1 kJ/mol per contact) that becomes significant over large complementary surfaces.
• Hydrophobic effect: nonpolar groups cluster to minimize disruption of water's hydrogen-bond network; entropy-driven (positive ΔS of released water).

C. The role of water and the dielectric

• Screening: the high dielectric constant of water reduces electrostatic energies roughly 80-fold versus vacuum.
• Consequence: the hydrophobic effect, not internal hydrogen bonds alone, provides the dominant driving force for protein and membrane assembly.

IV. Conformational Changes in DNA Molecules

Structural polymorphism of the double helix.

DNA is not a static rod; its helical geometry shifts with sequence, hydration and ionic environment.

A. The canonical B-form

• Watson–Crick pairing: A=T (2 H-bonds), G≡C (3 H-bonds); complementarity fixes the antiparallel duplex.
• Geometry: right-handed helix, ~10.5 base pairs per turn, ~3.4 Å rise per base pair, ~34 Å pitch.
• Grooves: wide major groove (protein-recognition site) and narrow minor groove.

B. Alternative conformations

The helix adopts distinct forms under changing conditions:

1. A-DNA: right-handed but compact (11 bp/turn, ~2.9 Å rise); favored at low hydration and adopted by RNA-DNA hybrids.
2. Z-DNA: left-handed, zig-zag backbone (12 bp/turn); favored by alternating purine-pyrimidine sequences (e.g. GC repeats) and high salt.

C. Denaturation and melting

• Melting temperature Tₘ: the temperature at which half the duplex is single-stranded, monitored by the hyperchromic rise in A₂₆₀.
• Sequence dependence: GC-rich DNA has a higher Tₘ because G≡C contributes three hydrogen bonds versus two for A=T.
• Reversibility: slow cooling allows reannealing, the physical basis of hybridization.

D. Supercoiling and topology

• Definition: over- or under-winding of the double helix, quantified by linking number Lk = Tw + Wr (twist + writhe).
• Biological role: negative supercoiling stores torsional energy that aids strand separation; controlled by topoisomerases.

V. From DNA to RNA

Transcription—copying genetic information into a working transcript.

Transcription synthesizes an RNA copy of a DNA template, the first step in gene expression.

A. Purpose and principle

• Template reading: RNA polymerase reads the template strand 3′→5′ and builds RNA 5′→3′.
• Base substitution: uracil (U) replaces thymine (T); the RNA sequence matches the coding strand with U for T.
• No primer required: unlike DNA replication, RNA polymerase initiates de novo.

B. The three stages

1. Initiation: polymerase binds a promoter (e.g. the −10 TATA-box region in bacteria), unwinds ~14 bp, forming the open complex.
2. Elongation: nucleotides added via nucleophilic attack of the 3′-OH on the incoming NTP's α-phosphate, releasing pyrophosphate.
3. Termination: release triggered by a hairpin plus U-run (intrinsic) or by a terminator protein (Rho-dependent).

C. Energetics and fidelity

• Driving force: hydrolysis of the released pyrophosphate (PPᵢ → 2 Pᵢ) makes chain growth thermodynamically irreversible.
• Error rate: ~1 in 10⁴–10⁵, higher than replication because most transcripts are transient.

D. Eukaryotic processing

• 5′ cap: 7-methylguanosine added to protect and mark the transcript.
• Splicing: introns excised, exons joined by the spliceosome.
• Poly-A tail: ~200 adenines added at the 3′ end for stability and export.

VI. The Biophysics of RNA

Structure, folding and dynamics of a single-stranded functional polymer.

RNA combines information storage with the ability to fold into catalytic and regulatory shapes, making its physics richer than that of DNA.

A. Chemical distinctions from DNA

• 2′-OH group: ribose bears a hydroxyl at C2′, enabling additional hydrogen bonding but making the backbone base-labile.
• Single-stranded default: RNA folds back on itself rather than pairing with a complementary strand.
• A-form helices: intramolecular duplexes adopt the compact A-geometry.

B. Secondary structure elements

Base pairing within one strand creates recurring motifs:

• Hairpin/stem-loop: a self-complementary stem capped by an unpaired loop.
• Bulges and internal loops: unpaired residues interrupting a stem, introducing flexibility.
• Wobble pairing: non-canonical G·U pair (2 H-bonds) extends folding and codon options.

C. Tertiary folding and thermodynamics

• Pseudoknots and coaxial stacking: long-range contacts pack helices into a compact fold.
• Folding stability: governed by base-stacking enthalpy and by counterion (Mg²⁺) binding that neutralizes backbone phosphate repulsion.
• Free-energy accounting: overall ΔG_fold is estimated by summing nearest-neighbour stacking and loop-penalty terms.

D. Functional consequences

1. Ribozymes: RNA that catalyzes reactions (e.g. the peptidyl transferase center of the ribosome, self-splicing introns), showing that a folded RNA active site can rival protein enzymes.
2. Regulatory RNAs: riboswitches change conformation on ligand binding to switch genes on or off, and tRNA's cloverleaf-into-L-shape fold positions the anticodon and amino acid for translation.

E. Significance in the RNA-world context

• Dual capacity: because RNA both stores information and catalyzes reactions, it is proposed as the primordial molecule bridging chemistry and biology.
• Biophysical lesson: the same weak-interaction hierarchy that folds proteins folds RNA, unifying the physics of all biomolecules under one energetic framework.