Biochemistry basics

• BIO515 Exam 1 Review covers major concept of biomolecules from Lehninger 7th Edition.

Biomolecules and Key Chemical Groups

• Biological macromolecules are long chains with freely rotating single bonds, allowing conformational flexibility.
• Macromolecules can form infinite yet specific conformations.
• Non-covalent interactions allow macromolecules to reversibly bind ligands.
• Macromolecular structures determine their function.
• Ligands bind using shape and charge complementarity via weak interactions like hydrogen or ionic bonding.
• Carbon, oxygen, hydrogen, and nitrogen form the basis of cellular life.
• Covalent bond types and configurations determine molecular shape.
• Noncovalent weak interactions are important for the shape of larger molecules.

Lysozymes and Covalent Bonds

• Lysozymes in tears and egg whites kill bacteria by matching the shape of the molecule to bacterial cell walls.
• Specific atoms break bacterial cell wall covalent bonds.
• Covalent bonds involve sharing electrons (strongest interaction).
• Valence electron, 2 electrons come from each atom.
• Bond polarity depends on involved atoms.
• Nonpolar bonds include C-H and C-C.
• Polar bonds include O-H, N-H, C-O, and C-N, resulting in partial charges.
• Covalent bonds form the backbone of all major macromolecules.
• Single bonds rotate, but double bonds do not.

Ionic and Hydrogen Bonds

• Ionic bonds involve the attraction of opposite charges (+,-).
• Salt bridges are an example of ionic bonds.
• Attraction is stronger when ions get closer.
• Hydrogen bonds involve sharing a hydrogen atom between a hydrogen acceptor and donor.
• O-H and N-H groups have highly electronegative O or N atoms that lead to partial positive charge formation.
• Oxygen in polar covalent bonds (C-O, C=O) serve as hydrogen acceptors.

Hydrophobic Interactions and Thermodynamics

• Hydrophobic interaction involves nonpolar substances in polar substances (especially water).
• Hydrophobic effect is the preference for nonpolar molecular surfaces to interact and displace water.
• Lipids disperse in solution as a result.
• Nonpolar lipid tails surrounded by ordered water molecules decrease entropy.
• Micelles sequester hydrophobic groups from water, minimizing ordered water and maximizing entropy.
• The hydrophobic effect favors ligand binding, and stabilizes enzyme-substrate interactions.
• Binding sites in enzymes and receptors are often hydrophobic.
• These sites can bind steroid hormones and contribute to protein folding and stability.
• Van der Waals interactions involve electrons of nonpolar substances (stacking).

Thermodynamics and Reactions

• Gibbs free energy is available to do work: ΔG= ΔH-TΔS.
• Enthalpy (H) describes the numbers/kinds of bonds.
• Temperature is denoted by T.
• Entropy (S) describes randomness/disorder.
• A negative ΔG means reaction is favored/spontaneous.
• A positive ΔG means reaction is not favored/nonspontaneous.
• ΔG is usually negative if entropy increases.
• Exergonic reactions release energy, LESS energy in products than reactants (e.g., hydrolysis of ATP).
• Endergonic reactions require INPUT of energy. MORE energy in products than reactants (e.g., photosynthesis).
• OIL RIG: Oxidation is losing electrons; Reduction is gaining electrons.
• Combustion has a favorable -ΔG, releasing energy.
• Cellular respiration increase in molecules and is favorable and -ΔG.

Chapter 2: Water Properties

• Water dissolves substances by forming a sphere of hydration, with many H-bonds around the substance.
• Crystal lattice gets reduced during dissolution.
• Water molecules in bulk have high entropy, can rotate, and form H-bonds with other molecules.
• Water around lipids has less freedom, forms a static layer, and reduces entropy.
• Hydrogen bonds give water its cohesiveness, adhesiveness, and high heat of vaporization.
• Liquid water is highly mobile, constantly forming/breaking bonds.
• Individual H-bonds weak, but together they're strong.
• Water forms hydrogen bonds with polar solutes (N-H, C-N, O-H, C-O bonds).
• Water is a good solvent for hydrophilic charged and polar substances like amino acids, peptides, and carbohydrates.
• Water is a poor solvent for hydrophobic nonpolar substances.
• Hydrophobic substances include aromatic moieties, aliphatic chains, and nonpolar gases.
• Osmosis is the movement of free water across a semi-permeable membrane.
• Water moves towards the side with more solute.
• Cells adapt to osmotic pressure.
• Osmolarity depends on the number of solute molecules (moles), not mass

Ionization and pH

• Water can ionize into a proton (H+) and a hydroxide ion (OH-).
• Water ionization is a rapid, reversible process.
• The equilibrium is strongly leftward.
• The equation for constant Keq = [H+][OH-]/[H2O].
• Ionic product of water = 1*10^-14 M
• pH is the negative log of H+ concentration: pH= -log[H+] = log(1/[H+]).
• A 1 pH unit change means a 10-fold change in H+ concentration; 2 pH units is 100-fold change.
• Biological molecules are pH-sensitive because structure and activity are affected by pH changes.
• Acids produce H+ in aqueous solutions, release H, and are proton donors/electron pair acceptors; H+ is a strong acid.
• Bases produce OH- in aqueous solutions, release OH, and are proton acceptors/electron pair donors; NaOH is a very strong base.
• Weak acids and bases don't fully ionize.
• Dissociation depends on the acid dissociation constant Ka.
• pKa=-log Ka
• Low pKa = stronger acid.
• High Ka shifts equilibrium to the right = stronger acid.
• pH Equation is pH=pka+log([A-]/[HA]), where A-= proton acceptor and HA= proton donor.
• At pHs below pKa, HA>>A-.
• At pHs above pKa, A->>HA.

Buffering

• Buffers resist pH change.
• At pH=pKA, there's a 50:50 mixture of acid and conjugate base forms.
• Buffering capacity lowers when pH differs from pKa by or more than 1 pH unit.
• When H+ or OH- is added near pKA, the buffered solution captures the H+in its equilibrium.
• Buffering continues until all weak acid or its conjugate base is used up and buffering power is lost.

Buffer Systems

• Intracellular pH maintenance is essential for cells.
• Cells use phosphate, bicarbonate, and histone as buffer systems.
• Labs use sulfonic acids of cyclic or primary amines as buffer systems.

Chapter 3: Amino Acids, Peptides, and Proteins

• All amino acids contain an amino and a carboxylic group (-COOH), acidic once releases a proton to become -COO.
• Characteristics unique to each amino acid give it its properties.
• Proline is the only AA where the R group connects to backbone
• All AA are chiral, except glycine.
• Nonpolar groups favor hydrophobic interactions with each other or with ligands and tend to cluster inside proteins (away from water).
• G and P are alpha helix breakers where a-helix end or turn around.
• M is the starting amino acid of every protein that is initially translated.
• Aromatic R Groups are non polar and hydrophobic.
• F is more hydrophobic than Y and W.
• Often form stacking interactions with each other or with NA.
• Y is a site of phosphorylation at the -OH.
• Polar uncharged R groups can form hydrogen bonds.
• Cysteine can form disulfide bonds.
• Negatively charged groups are weak acids having side chain pKa ~4.
• Positively charged groups have ionizable side chains, are basic.
• H pKa is near pH 7 that signifies at physiological.
• pH histidine side chain is partially pronated.

Hydropathy, Isoelectric Point and Protein Studies

• Hydropathy indicates if amino acid can interact with water (protein surface).
• Negative -G means favorable interaction (exterior).
• Negative -deltaG means unfavorable interaction (hydrophobic) (interior).
• Proline, glycine, tyrosine, tryptophan, Serine, threonine, asparagine glutamine, lysine, histidine, aspartate, glutamate get located at surface.
• Isoelectric Point (pI) is the pH where the molecule is electrically neutral (no net charge).
• With non-ionizable side chains the Isoelectric Point (equivalence point, pI) is the average of the pKas of carboxy and amino groups.
• pI = pK1+pK2/2
• Deprotanated = -1
• Protanated = +1
• When the pH matches the AA pI, the aa is least soluble in water, aa does not migrate in electric field, same is true for peptides and proteins.
• Peptides are condensation products of amino acids called residues.
• The start is N-terminus, the end is C-terminus.
• To study a protein one must purify it.

Protein Purification Steps

• Breaking cells/homogenization/extraction with a blender, osmotic shock, sonicator, glass beads, enzymatic treatment.
• Differential centrifugation separates cell components by size/density.
• Larger sediments form pellet on the bottom of the tube while the smaller components remain in suspension above (supernatant).
• Salting-out/dialysis and concentration causes hydrophobic aggregation with unfolded proteins or by removing the water to make protein stick together.
  • (a) Solvation is certain ions required for the protein to be soluble ("salting in")..
  • (b) Aggregation involves certain salts disrupting water solvent shell and proteins precipitate ('salting out”).
  • (c) Hydrophobic aggregation increases as solution pH nears protein pI. Proteins tend to be less soluble at pI because the protein molecule's net neutral charge aggregates without repelling each other.
• Ion-exchange occurs at pHs BELOW the pI that causes the protein gains protons (+ charge). pHs ABOVE the pI will cause protein to lose protons (- charge).
  • (a) Two ion exchange types-
    • (i) Anion where (-) solid phase is (+) charged binds negatively charged proteins.
    • (ii) Cation where (+) solid phase is (-) charged so it binds positively charged proteins.
  • (b) To elute proteins either change the salt concentration (salt ions bind to the protein neutralizing charge) or change the pH because pH is a factor (depends on a proteins pl).
• Affinity chromatography interactions with covalently bound ligands depending on interact intensity. A too-high affinity ligand results to purification is ineffective.
• Column chromatography means the solid phase remains fixed, and the mobile part mix moves through.
• Gel-filtration chromatography/ Size exclusion means smaller molecules take longer to travel because they get stuck inside the beads, so larger ones move past first.
• SDS-PAGE (also isoelectric focusing and 2D PAGE) involves samples atop pores with small molecules move quickly and separated by size using gelk.
  • (a) SDS binds proteins and provides of proteins a charge, (MW ~charge/mass ratio), denatures and makes easier for gel migration.
  • (b) SDS-PAGE helps Purity of protein smaple, confirm MW of protein of interest.
• Functional assay in final purification stages measure Lysozyome activity by purified protein sample on paper disk on bacterial petri dish is tested by inhibitation of bacterial cell death (lysis" halo).
• Mass spec uses methods to determine and identify proteins by MW using minute material amounts. MALDI-MS for measuring proteins, (MS/MS) to sequence and for short peptide. Often find unknown proteins.
• Workflows implement Affinity chromatography to separate size and charge.

Amino Acid Homology

• • means Invariant conserved aa.
• : means conservative substitutions (same aa family).
• No symbol means nonconservative substitutions (diff aa family).

Chapter 4: Three-Dimensional Structures of Proteins

• Primary structure is the linear sequence of amino-acids in a peptide/protein (e.g., -Ala-Glu-Val-Asp).
• Secondary structure are alpha-helix and Beta sheet.
• Tertiary structure: Combination of both secondary structures/ elements.

Protein Interactions

• Overall, weak interactions within protein structures collectively contribute to the stability, conformational flexibility, and function of proteins.
• Promote folding in natives structures, interacting and executing biological cellular functions. molecules.
• Peptide bond removes + and – charges from the functional groups with rigid and planar characteristics
• Carbonyl oxygen has a partial negative charge, and the amide nitrogen a partial positive. Small electric dipole formation.
• All peptide bonds occur in this trans configuration, peptide bonds = rigid
• Rotation occurs in single bonds around the peptide.

Angles of Proteins

• Phi angle- around C-N-Ca-C.
• Psi angle- around N-Ca-C-N.
• Secondary- local arrangement of the polypeptide backbone.
• Hydrogen bonding of backbone not chain forms Structure thru C=O and N-H (H-Bonds).
• Backbone N-H is H bond donor + C=O is H bond acceptor (except Prolines containing N).
• Alpha-helix shape happens with stabilize hydrogen bonds of nearby atoms/groups.
• Residues of C=0 and N-H backbone coils.

α-Helix Properties

• Various amino acids have different properties in helices
• R-group interactions can stabilize or destabilize an α-helix (3-4 AAs apart).
• α-helix has a dipole, affecting certain end amino acids
• Amphipathic helices are either polar or nonpolar ,or faces with polar side chains
• Alanine aa is likely in alpha helices because of its small R group
• Proline destabilizes α-helices with inflexible diheral angles and no H to contribute to H-bond.
• Glycine doesnt have a chain is too flexible.
• Charged Aas are located on the N + C terminals

β-Sheets Characteristics

• Chain stretches of AAs that pair parallel form sheet shape
• Backbone atoms from adjacent chain segmenstabilize H-bonds.
• "Pleated" from planar geometry from around the partially bond
• Alternate side chain is above or below sheets.
• Parallel β sheets have bent or weaker H-bonded strands in the same direction .
• Antiparallel β sheets have stronger H-bonded strands run.
• The B turn: A 4 residue stretch induces a sharp hairpin turn when connects anti-parallel beta-sheets and/or alpha-helices containing Usually proline and glycine
• First and 4th aa make hydrogen bond.
• Random coil: No particular 2° Structure- irregular arrangement of the polypeptide chain.
• Dihedral angles and Ramachandran plots: Ramachandran plots give distribution for angles in a protein.

Tertiary and Quaternary Structures

• Tertiary (or globule) refers to proteins with overall spatial arrangements.
• Multiple Secondary structures element (α-helices, B sheets).
• Stabilize interaction for aa side chains
• Disulfide bridges
  • (a) Globular: Majority of proteins folded in shapes (myoglobin, or hemoglobin).
  • (b) Fibrous: Polypeptides in keratin proteins
  • Secondary structures are H-bonding of H peptide backbone while tertiary bonds uses non covalent types (i.e. disulfide covalent bonds).
• Quaternary is proteins complexes from bound polypeptides . Tetramer-containing protein.

Motifs to Crystalography

• Motif: secondary arrangements (alpha all, beta all or both) in different protein motif.
• Domain structure: polypeptide chains folding as independent X-ray crystallography
  • purify protein, crystallize protein, collect diffraction, calculate electron density, fit residues into density
  • high quality (< 3 Angstroms) - Low quality (> 5 Angstroms).
• NMR: Purify protein/dissolve, collect NMR data, assign signals/can't crytallize. Can get insight.
• Cryo-EM uses electron passes through the proteins . Reveals difficult proteins.
• Monomers have single polypeptide ,example insultin.
• Dimers have 2 identical/non identical subunits via hbond
• Trimer contains 3 units. Example is collagen.
• Tetramer has 4 (hemoglobin).

Protein Folding & Control

• Protein unfolding, denaturation means without primary structure. Possible denaturants from chaotropic → H2O change.
• Denaturation all at once for cooperative process (destabilizing losses in part).
• Protein folding helps fold linear polypeptide with functionalities/charactersitic.
• Folding experiment via RNase revealed the aa determines structure which denaturant has folded protein to.
• Levinthal paradox says native structures takes more time
• Drive protein folding via formation of localized peptide and sticking (low energy).
• Chaperones binding to stabilize folded chains folding within the cytoplasm.
• GroEL-GroES mechanism unfold proteins for another folding chance
• Protein Misfolding creates conformations during syntheis mutation
• Misfolded proteins cause amyloid fibril diseases.

Prions and Homeostasis

• Prions cause other proteins to misfold, causing Kuru.
• Proteostasis- Protein folding homeostasis to quality control in cells.

Test your knowledge of biochemistry! This quiz covers buffer systems, amino acids, protein structure, and separation techniques, and their chemical properties. Perfect for students studying biochemistry or related fields.