Protein Structure and Function - Vocabulary Flashcards

Overview

  • Proteins are the most diverse macromolecules in terms of structure and function.
  • A protein’s three-dimensional structure is called its conformation.
  • Correct folding is critical for function; function is derived from structure.
  • Chapters cover from primary sequence to quaternary assemblies, plus regulation, folding, motifs, domains, and methods to study proteins.

Monomers, Polymers, and Key Terms

  • Amino acid: the building block with structure shown (general form includes H, carboxyl, amino, and unique side chain R).
  • Peptide bond: linkage between amino acids; peptide bonds are planar due to partial double-bond character of the C–N bond.
  • Monomer to polymer:
    • Amino acid → dipeptide → tripeptide → peptide (2–30 residues) → polypeptide (longer chain, up to ~4000+) → protein (a polypeptide folded into a specific conformation).
  • Memorize amino acids and their side chains (R groups) for understanding folding and interactions.
  • In diagrams: peptide bond forms between carbonyl carbon of one amino acid and amine nitrogen of the next, producing a repeating backbone with side chains (R groups) protruding outward.

Peptide Bonds and Connectivity (Fig. 3-3 variants)

  • Peptide bonds are planar because the C–N bond has partial double-bond character.
  • The carbonyl oxygen and amide nitrogen contribute to the stability and planarity of the peptide bond.
  • In short peptides, early examples show amide linkage between adjacent amino acids forming the backbone.

Key Terminology (Page 4)

  • peptide: short chain of amino acids (2–30 residues).
  • polypeptide: longer chain (up to 4000+ residues).
  • protein: a polypeptide that has folded into a specific conformation.

Levels of Protein Structure

1) Primary structure
  • Linear sequence of amino acids.
  • The specific sequence contains all intrinsic information governing folding into the native 3D structure.
  • Example sequence: Ala-Glu-Val-Thr-Asp-Pro-Gly-
  • Primary structure is depicted as a linear chain with N-terminus and C-terminus.
2) Secondary structure
  • Local folding patterns: alpha (α) helix, beta (β) strand, reverse (β) turns, random coil.
  • α-helix: right-handed coil stabilized by intramolecular hydrogen bonds between backbone amide N–H and carbonyl O of the amino acid four residues earlier (i to i+4).
  • β-sheet: formed by β-strands connected laterally by at least two or more backbone hydrogen bonds; strands can be parallel or antiparallel.
  • Random coil: unstructured regions connecting helices and sheets.
  • Roughly 60% of an average protein’s 3D structure is due to secondary structure elements.
  • Notation in figures uses α and β; turns are often displayed as ẞ turns.
3) Tertiary structure
  • Overall folding of the polypeptide chain into a single 3D structure (a full domain or multiple domains).
  • Key interactions driving folding:
    • Hydrophobic interactions (driving burying of nonpolar side chains in the core).
    • Hydrogen bonds (within backbone and sometimes side chains).
    • Ionic (electrostatic) interactions.
    • van der Waals interactions.
    • Disulfide bonds (covalent; sometimes present) between cysteine residues.
  • Noncovalent interactions are typically dominant; covalent disulfide bonds are optional depending on protein.
4) Quaternary structure
  • The assembly of multiple polypeptide chains (subunits) into a functional protein complex.
  • Interactions are similar to those that drive tertiary structure (noncovalent and sometimes covalent).
  • Example: a trimeric protein consisting of three subunits (HA1/HA2 in hemagglutinin).
  • Proteins can be monomeric (one subunit) or multimeric (>1 subunit).
Summary Figure (Fig. 3-1a, 3-1b)
  • Structural levels: Primary → Secondary → Tertiary → Quaternary; these levels can be viewed alongside the concept that regulation and function arise from structure and conformation.

Protein Structure Details and Visuals

  • Secondary structures can be visualized by backbone traces, ball-and-stick, ribbons, or surface plots.
  • α-helix specifics:
    • 3.6 residues per turn; typical α-helix has a macrodipole because carbonyl oxygens point down and amide nitrogens point up, creating a dipole along the helix axis.
    • R groups project from the helix sides; one side can be polar/charged and the opposite hydrophobic (amphipathic helix).
  • β-sheet specifics:
    • 5–8 amino acids per strand; hydrogen bonds between backbone amide and carbonyl groups hold strands together, forming a pleated sheet.
    • Strands can be parallel or antiparallel; side chains project above and below the sheet.
  • Reverse turns (β turns): typically 4 amino acids forming a U-turn; glycine and proline are common in turns (special amino acids or helix breakers).
  • Random coils/loops: connect helices and β-sheets; lack defined structure but can be functionally important.
  • Amphipathic helices: hydrophobic and polar faces; used in interactions and membranes (e.g., Leucine zippers).

Protein Motifs and Domains (Protein Structural Motifs)

  • Motif: a region containing various secondary structures with a defined 3D topology and a function; motifs recur in different proteins.
  • Domain: larger, functional modules that may be composed of one or more motifs; domains can be structural, functional, or topological.
  • Four main domain classes:
    • Functional domain: catalytic, DNA-binding, RNA-binding, ligand-binding, Ca2+-binding, etc.
    • Structural domain: collection of secondary structures folded into a distinct structure (often at least ~40 amino acids).
    • Topological domain: membrane-related partitions (extracellular, cytosolic, membrane-spanning regions).
    • Domain definitions can be based on enrichment of specific amino acids (acidic, basic, hydrophobic, etc.).
  • Examples of motifs:
    • Coiled-coil motif (leucine zipper): amphipathic helices, forming dimers; Leu every 7th residue; often used in DNA-binding proteins.
    • EF-hand/Helix-loop-helix (HLH) motif: Ca2+-binding regulatory proteins; contains Ca2+-binding loops and helices.
    • Zinc-finger motif: often DNA-binding; consists of Cys and His coordinating Zn2+.

Hemagglutinin Example and Domain Modularity

  • Hemagglutinin (HA) comprises distal globular domain and proximal fibrous domain; HA1 and HA2 subunits form a trimer in the viral membrane.
  • Protein domains are modular and can be found in different proteins; cleaved domains can still fold and function.
  • EGF precursor, integrins, and other proteins illustrate how domains (modules) can be combined in different proteins to achieve diverse functions.

Protein Folding Principles

  • The Hydrophobic Effect (oil-drop model): hydrophobic side chains cluster in the protein core; water molecules reorganize to minimize exposed hydrophobic surface, driving folding and stability.
  • Folding is thermodynamically favorable through multiple steps: secondary structures form first, followed by domain formation, then final tertiary structure.
  • There are many potential folding pathways, but a native state is usually reached as the most thermodynamically stable conformation.
  • Number of possible folds grows geometrically with chain length (e.g., a protein with 100 AAs could theoretically fold in up to 2×10902 \times 10^{90} ways), yet real proteins fold into a single native state.
  • Folding is aided by cellular factors:
    • Molecular chaperones (e.g., Hsp70, Hsp90) stabilize unfolded/partially folded proteins and assist folding using ATP cycles.
    • Chaperonins (GroEL/GroES in bacteria; TRiC in eukaryotes) form barrel-like chambers that provide an isolated folding environment.
  • Disulfide bonds can stabilize folding by covalently linking cysteine residues.
  • Proline isomerases (cis-trans isomerases) catalyze cis/trans isomerization at X-Pro peptide bonds, influencing folding kinetics.
  • Denaturation/renaturation experiments show that many proteins can refold, but some do not renature easily; denaturing agents include urea, guanidine HCl, salt, and pH changes.

Protein Misfolding and Disease

  • Abnormally folded proteins can aggregate into amyloid fibrils and plaques, contributing to diseases such as Alzheimer's, Parkinson's, prion diseases, and various systemic amyloidoses.
  • Misfolded proteins are normally degraded, but accumulation can lead to pathology.
  • The aggregation often involves cross-β sheet architectures in amyloids.

Functional Design of Proteins

  • Ligand: a molecule that binds a protein with high specificity (lock-and-key analogy).
  • Substrate: reactant that binds an enzyme.
  • Binding can induce conformational changes (induced fit) that optimize catalysis or signaling.
  • Affinity: strength of protein-ligand binding; quantified by equilibrium constant Keq or dissociation constant Kd (lower Kd = higher affinity).
  • Specificity: ability to distinguish between ligands; designed by complementary shapes and chemistries.
  • Enzymes act by stabilizing the transition state, thereby lowering activation energy and increasing reaction rates.
  • Active site: region where substrate binds; catalytic site: region where chemical transformation occurs.
  • Example: Protein Kinase A (PKA) illustrates induced fit and domain movements in catalysis with a glycine lid and nucleotide-binding pocket; serine/threonine residues targeted by phosphorylation.
  • The concept: enzymes lower activation energy by binding the transition state (a pentavalent transition state with Mg2+ coordination is shown in the PKA example).

Enzyme Kinetics (Bare-bones minimum)

  • Km (Michaelis constant): a measure of substrate affinity; inversely related to affinity.
  • Vmax: maximum velocity at saturating substrate concentration; related to enzyme turnover number.
  • Turnover number (kcat): rate constant for substrate processing per enzyme molecule per second.
  • Michaelis-Menten equation (steady-state):
    v=V<em>max[S]K</em>m+[S].v = \frac{V<em>{max} [S]}{K</em>m + [S]}.
  • For multiple substrates, Km values differ between substrates; higher affinity corresponds to a lower Km for that substrate.

Regulation of Protein Function

  • Environmental regulation: pH affects enzyme activity (e.g., lysosomal enzymes optimum near pH ~5; cytosolic pH is higher).
  • Physical association of catalytic domains: scaffolding proteins can bring enzymes into proximity, enabling sequential reactions.
  • Multiple catalytic domains within one protein can enable coordinated catalysis.
  • Degradation and turnover:
    • Extracellular proteases (e.g., trypsin, chymotrypsin) and exopeptidases (aminopeptidases, carboxypeptidases).
    • Intracellular pathways: lysosomal degradation at low pH; ubiquitin-proteasome pathway in cytosol.
    • Ubiquitin pathway: ubiquitination tags proteins for proteasomal degradation via a cascade of E1, E2, and E3 enzymes; chains can also signal in regulation (e.g., signaling, immune functions).
  • Regulatory subunits vs catalytic subunits in protein function (e.g., PKA): regulatory subunits bind ligands (e.g., cAMP) and control catalytic subunits via conformational changes.
  • Allosteric (noncovalent cooperative) binding:
    • Example: PKA regulatory subunits cooperatively bind 4 cAMP molecules; hemoglobin cooperatively binds 4 O2 molecules.
    • Allosteric switches can turn activity on or off via ligand-induced conformational changes.
  • Allosteric switches examples:
    • Ca2+ binding to calmodulin (Ca2+-calmodulin activates target peptides via four Ca2+-binding EF-hand motifs).
    • Guanine nucleotide-binding switch proteins (Ras, Ran) use GTPase cycling controlled by GEFs (guanine-nucleotide exchange factors) and GAPs (GTPase-activating proteins).
  • Post-translational covalent modifications:
    • Phosphorylation: reversible, typically on Tyr, Ser, or Thr; regulated by kinases and phosphatases.
    • Ubiquitination: covalent attachment of ubiquitin can signal degradation or regulatory roles; poly-ubiquitination often targets for proteasome; isopeptide bonds form between ubiquitin and lysine residues on target proteins.
    • Proteolytic cleavage/processing: proprotein processing to activate function (e.g., proinsulin to insulin; furin, PC2/PC3 proteases); regulated processing in the secretory pathway.
    • Other covalent modifications can alter function, localization, and interactions.
  • Important: post-translational modifications can regulate protein activity beyond degradation (signaling, localization, and complex formation).

Protein Degradation Pathways

  • Extracellular proteolysis: digestive proteases like trypsin and chymotrypsin; exopeptidases trim residues from ends of proteins.
  • Intracellular degradation:
    • Lysosomal pathway: hydrolytic enzymes operate in acidic environments (pH ~5).
    • Proteasome pathway: cytosolic degradation via ubiquitin tagging (E1, E2, E3 enzymes) leading to proteasomal proteolysis.
  • Ubiquitin details:
    • Ub is a 76-aa protein; ubiquitination involves E1 (activating), E2 (conjugating), E3 (ligase) enzymes.
    • Ub is recycled by deubiquitinases (DUBs) before degradation.
    • Destruction boxes (signal sequences) on target proteins direct ubiquitination.
  • Visual: Ub chains attach to lysines on targets; proteasome unfolds and digests into peptides.

Post-Translational Modifications and Processing (Examples)

  • Cleavage/Processing in the secretory pathway:
    • Prohormone processing (e.g., proinsulin to insulin) by proteases such as furin and PC family proteases; disulfide bonds can stabilize intermediates.
  • Intein splicing (self-splicing proteins): rare auto-catalytic events where an intron within a protein is excised; not likely to be on exams here.

Protein Purification and Analytical Techniques (Methods)

  • Purification principles: proteins differ by size, charge, solubility, stability, and binding affinity; purification exploits these properties.
  • Centrifugation:
    • Differential centrifugation separates by mass/density; larger particles pellet earlier; smaller particles remain in supernatant.
    • Rate-zonal centrifugation uses a density gradient (e.g., sucrose) to separate particles by mass as they migrate through gradient layers.
    • Sedimentation coefficient S (Svedberg units) depends on mass, shape, and density; used to estimate size and mass.
  • Equilibrium density-gradient centrifugation: separates organelles by density in a gradient; useful for organelle purification and DNA purification.
  • Electrophoresis:
    • SDS-PAGE denatures proteins and coats them with negative charge; separates by size as they migrate through polyacrylamide gel.
    • Two-dimensional gel electrophoresis combines IEF (isoelectric focusing) by pI in first dimension and SDS-PAGE by size in second dimension.
    • Isoelectric point (pI) is the pH at which a protein has no net charge.
  • Chromatography:
    • Gel filtration (size-exclusion): separates by size; larger proteins elute first.
    • Ion-exchange chromatography: separates by charge; depends on protein’s net charge and salt gradient for elution.
    • Affinity chromatography: exploits specific binding interactions (e.g., antibody–antigen; enzyme–inhibitor);
      eluted by changing pH or competing ligands.
  • Detection methods:
    • Western blotting (immunoblot): antibodies detect specific proteins after gel transfer.
    • Immunoprecipitation (IP) to pull down proteins and interactors for analysis.
  • Radioisotopes and labeling:
    • 32P, 35S, 125I, 14C, 3H used for tracking molecules; pulse-chase experiments study dynamics of synthesis, processing and turnover.
    • Pulse-chase example: use 35S-Met to label newly synthesized proteins briefly, then chase with unlabeled Met to observe maturation, trafficking, and degradation.
  • Protein sequencing and identification:
    • Edman degradation: sequential N-terminal amino acid identification via HPLC.
    • DNA sequencing and in silico translation to deduce protein sequence.
    • Mass spectrometry (MS): peptide mass fingerprint after proteolysis; MS/MS to sequence peptides; MALDI-TOF and ESI (electrospray) approaches.
    • 2D MS (MS/MS) can derive amino acid sequences from peptides.
  • In vitro peptide synthesis: short peptides (10–100 aa) for antibody production, substitution studies, and folding analyses; iterative protection/deprotection chemistry used to build sequences.

Protein Structure Determination Technologies

  • X-ray crystallography: determines precise 3D structure from diffraction patterns; requires crystals; Fourier transform converts diffraction spots into 3D structure.
  • Cryo-electron microscopy (cryo-EM): high-resolution structure from images of frozen-hydrated samples; suitable for large complexes.
  • Nuclear magnetic resonance (NMR): uses magnetic fields to determine distances and angles between atoms; best for smaller proteins/domains (< ~20 kDa).
  • The proteomics workflow often combines LC-MS/MS with database searching to identify proteins in complex samples.

Protein Families, Evolution, and Heme Cofactors

  • Proteins in a family share a common evolutionary ancestor; homologous proteins show high sequence/structure similarity across species.
  • Evolutionary trees illustrate divergence of related proteins (e.g., globins like hemoglobin and myoglobin across vertebrates and invertebrates).
  • Heme as a cofactor (prosthetic group) in hemoproteins (e.g., hemoglobin, myoglobin, leghemoglobin) providing oxygen binding capability.

Protein Domains and Modularity

  • Proteins are modular: domains are larger functional blocks that can be swapped or recombined across proteins.
  • A domain can be defined independently by its topology and function; folding of domains can be autonomous when isolated.
  • Examples: Epidermal Growth Factor (EGF) domains, Ig-like domains, transmembrane domains, Kringle domains, etc.
  • The modular design enables domain shuffling and creation of multidomain proteins with diverse functions.

Four Broad Structural Categories of Proteins (Ch. 3)

  • Globular proteins: roughly spherical, compact, often enzymes or transport proteins (e.g., myoglobin).
  • Fibrous proteins: elongated, structural roles (e.g., keratin, collagen).
  • Integral membrane proteins (IMPs): span membranes with hydrophobic regions (often α-helical in membranes).
  • Intrinsically disordered proteins: lack a single native state until bound; can be induced to fold by interactions; important in signaling and regulation; often involved in transient complexes.
  • Note: proteins can contain regions that belong to different categories (e.g., a single protein may have ordered domains and disordered segments).

Allosteric Regulation and Signaling

  • Allosteric regulation involves noncovalent cooperative binding that changes the protein’s conformation and activity.
  • Calmodulin model: Ca2+ binding to calmodulin leads to conformational changes that regulate target interactions; involves four EF-hand motifs.
  • Ras/Ran GTPases: regulatory switches controlled by GEFs and GAPs; bidirectional control by exchange of GDP for GTP and hydrolysis, not by phosphorylation alone.
  • Cyclic nucleotides (cAMP) and kinases: cAMP binding to regulatory subunits can activate catalytic subunits (as in PKA) via conformational shifts.
  • Allosteric switches allow proteins to respond to cellular signals with high cooperativity and precision.

Posttranslational Modifications: Broad Roles

  • Phosphorylation: reversible addition of phosphate groups by kinases; modulates activity, interactions, localization; often on Ser/Thr/Tyr.
  • Ubiquitination: tagging with Ub to signal degradation or regulatory roles; involves E1, E2, E3; can form chains; reversible by deubiquitinases.
  • Proteolytic processing: activation of proproteins (e.g., insulin, proinsulin); occurs via specific proteases in secretory pathways (furin, PC family).
  • Covalent modifications beyond ubiquitination: other isopeptide bond formations and covalent crosslinks that regulate function and interactions.

Key Takeaways: Structure Governs Function

  • The central thesis: structure determines function; proteins adopt conformations that enable specific binding, catalysis, signaling, and mechanical work.
  • Function emerges from the integrated behavior of primary sequence, secondary structures, domain organization, and quaternary assembly.
  • Stability and dynamics (breathing) allow conformational changes required for activity and regulation.

Quick Reference: Core Equations and Concepts

  • Michaelis–Menten kinetics (enzyme-c substrate reactions):
    v=V<em>max[S]K</em>m+[S]v = \frac{V<em>{max} [S]}{K</em>m + [S]}
  • Km reflects affinity: lower Km indicates higher affinity for substrate.
  • v at saturating substrate equals Vmax.
  • 3.6 residues per turn in an α-helix; take note for helices and dipole considerations.
  • Sedimentation coefficient S and its relevance to protein size and shape in centrifugation.

Connecting to Practice and Real-World Relevance

  • Enzymes as catalysts: lower activation energy by stabilizing transition states; relevance to drug design and biotechnology.
  • Protein purification and proteomics: isolating and identifying proteins from complex mixtures to study function, interactions, and disease mechanisms.
  • Structural determination techniques enable drug design, understanding disease mutations, and engineering proteins with novel functions.
  • Misfolding and amyloids are central to several neurodegenerative and systemic diseases; understanding folding pathways informs therapeutic strategies.

Summary: Hierarchy and Connectivity

  • Amino acids → primary structure → local secondary structures → tertiary structure (domain formation) → quaternary structure (multimeric assemblies) → function and regulation.
  • Modular design and domains enable functional diversity and evolutionary plasticity.
  • Regulation occurs at multiple levels: conformational changes, allosteric binding, covalent modifications, and controlled degradation.
  • Analytical and purification techniques allow the isolation, characterization, and identification of proteins within complex biological systems.