Comprehensive Protein Biochemistry Notes (9/5)

Protein structure, composition, and basic terms

  • Amino acids link to form polypeptides; example chains mentioned: amino acids (Amino acid 1, amino acid 2, …, AAN) progressing to a polypeptide with one peptide bond; example given: cytochrome c.
  • Many enzymes exist as multiple polypeptides; e.g., Hexokinase many isoforms. Mentioned concept: alpha2, alpha4, etc., leading to multimers or hetero-oligomers.
  • Definitions:
    • Homomeric protein: two or more identical polypeptides.
    • Heteromeric protein: two or more different polypeptides.
  • Notation for a quaternary composition example: two alpha and two beta polypeptides could form a heterotetramer α2β2. The note R suggests such compositions can be hydrolytic (enzymatic activity may be present).
  • General statement: composition-based naming can specify how many of each polypeptide are present in a multimeric protein.
  • Important takeaway: proteins can be monomeric or multi-subunit complexes with identical or different subunits; function often depends on the subunit composition and arrangement.

Molecular weight and charge from amino-acid composition

  • Key idea: you can estimate molecular weight (MW) from amino-acid composition if you know how many residues of each type exist.
  • Example setup (as presented in the transcript): a protein with subunits having given numbers of amino acids per subunit (e.g., α with 120 AAs, β with 100 AAs, γ with 50 AAs, δ with 48 AAs). The total MW is the sum of the masses of all residues minus the mass of water removed during peptide bond formation.
  • General formula for MW from composition:
    {
    m MW}{protein} \approx \sumi ni \, MW{residue,i} - (N-1) \, 18.01528,
    where $ni$ is the number of residues of type $i$, $MW{residue,i}$ is the average mass of residue $i$, and $N$ is the total number of amino acids (the number of peptide bonds is $N-1$).
  • An explicit example workflow from the transcript (conceptual, numbers illustrative):
    • Given composition by residue type (e.g., α, β, γ, δ) with each type’s number of residues and their counts of specific charged residues (R, K, D, E) and others.
    • Compute total residues: $N = N{ m α} + N{
      m β} + N{ m γ} + N{
      m δ}$.
    • Compute total number of basic residues: $N_{R+K} = ( ext{R count in α}) + ( ext{R count in β}) + ( ext{R count in γ}) + ( ext{R count in δ}) + ext{K counts likewise}$.
    • Compute total number of acidic residues: $N_{D+E}$ similarly.
    • Net charge approximation (at a given pH) will depend on these counts and terminal groups (see below).
  • Practical note: in the transcript an explicit, messy worked example was shown with multiple subunits and specific counts for R/K and D/E to illustrate how to accumulate a net RNK and a net D/ E; results were around a net positive charge, illustrating how charge is tallied from residue composition.
  • After calculating MW from the residue composition, the instructor also notes you can estimate the net charge at a specified pH by counting charged residues and accounting for terminal groups.

Charge calculation and pH dependence (RNKs vs D/E)

  • Core principle stated: RNKs (arginine and lysine) carry positive charges; D/E (aspartate and glutamate) carry negative charges. The charge balance changes with pH according to pKa values of ionizable groups.
  • Practical approach described:
    • Count the total number of arginines and lysines (RNK").
    • Count the total number of aspartates and glutamates (D/E).
    • For a rough estimate at physiological pH, the lecture suggests: Net charge ≈ (#R + #K) − (#D + #E) with additional contributions from the N-terminus and C-terminus depending on pH.
  • Terminus contributions (illustrative):
    • N-terminus typically carries a +1 charge at low to moderate pH; often still +1 around physiological pH (depending on local environment and pKa ~8).
    • C-terminus typically carries a −1 charge at physiological pH (pKa ~3–4 for the C-terminal carboxyl group).
    • At physiological pH (~7.4), a common simplifying assumption is that terminal charges roughly cancel, leading to Q ≈ (#R + #K) − (#D + #E). Note: actual values depend on pKa of specific termini and local microenvironment.
  • Summary formula (textual):
    • Net charge at pH is determined by the sum of the charges of ionizable side chains plus the termini; rough estimate used in the lecture is
      Q(pH)≈N<em>R+N</em>K−N<em>D−N</em>E +q<em>N−term(pH)+q</em>C−term(pH),Q(pH) \approx N<em>{R} + N</em>{K} - N<em>{D} - N</em>{E} \,+ q<em>{N-term}(pH) + q</em>{C-term}(pH),
      where $q{N-term}$ and $q{C-term}$ are the charges of the N- and C- termini at the given pH.
  • Takeaway: calculating the charge requires residue counts and awareness of pH effects; the lecture emphasizes “RNKs positive, D/E negative” as the key idea, with terminal charges as a corrective factor.

Protein synthesis, secretory pathway, and localization

  • ER and Golgi involvement:
    • Rough ER and smooth ER are involved in protein processing; Golgi apparatus performs “fine tuning” for correct cellular localization and function.
    • After processing, proteins are secreted into the cytoplasm and then localized to their intended compartments.
  • Concept of cellular localization is introduced, implying that post-translational processing affects where a protein ends up and how it functions inside the cell.

Protein phosphorylation and kinases

  • Phosphorylation targets amino acid side chains: serine, threonine, tyrosine.
  • Phosphorylation basics:
    • A kinase transfers a phosphate group from ATP to a hydroxyl group on Ser/Thr/Tyr.
    • Typical residues and functional groups involved:
    • Serine: –CH2–OH becomes –CH2–O–PO3^2−
    • Threonine: –CH(OH)–CH3 becomes –CH(OPO3^2−)–CH3
    • Tyrosine: phenolic –OH becomes –O–PO3^2− on the aromatic ring
    • Kinases are highly specific for the residue they phosphorylate (serine-specific kinases, threonine-specific, tyrosine-specific; multiple kinases can act on a single type but with different recognition motifs).
  • Phosphorylation reaction (schematic):
    ext{Protein{-}OH} + ext{ATP} \xrightarrow{\text{Kinase}, \,\mathrm{Mg^{2+}}} ext{Protein{-}OPO_3^{2-}} + ext{ADP}
  • Consequences of phosphorylation:
    • Acts as a molecular switch in signaling pathways (phosphoproteins).
    • Provides docking sites for adaptor proteins such as GRB2 that contain SH2 domains recognizing phosphotyrosine motifs.
  • Example signaling cascade (overview):
    • Growth factor binds receptor tyrosine kinase (RTK) receptor on the cell surface.
    • Receptor dimerizes and autophosphorylates tyrosine residues (pY).
    • pY recruits GRB2 (adapter protein); GRB2 binds SOS (a guanine nucleotide exchange factor).
    • SOS activates RAS by exchanging GDP for GTP on RAS (membrane-bound GTPase).
    • RAS-GTP activates RAF kinases (MAPKKK).
    • RAF phosphorylates MEK (MAPKK).
    • MEK phosphorylates ERK (MAPK).
    • ERK translocates to the nucleus, partners with transcription factors to drive gene expression (growth-promoting genes).
    • Result: progression of cell cycle and, in many contexts, cell proliferation.
  • Emphasis: phosphorylation is central to cell signaling; it allows communication from the cell surface to intracellular machinery via a cascade of kinase activations.

Receptor tyrosine kinase signaling cascade and cell fate decisions

  • RTK example pathway (as described):
    • Growth factor binds RTK → RTK dimerization → tyrosine phosphorylation (pY).
    • pY recruits GRB2 (adaptor) → GRB2 associates with SOS.
    • SOS activates RAS by GTP loading (RAS-GTP).
    • RAS-GTP activates RAF kinases (MAPKKK) → MEK (MAPKK) → ERK (MAPK).
    • ERK (phosphorylated form, often p-ERK) enters the nucleus and, with other factors, drives transcription of growth-promoting genes.
  • Consequences:
    • Increased mRNA production for growth-related proteins.
    • One cell division event can follow (cell cycle progression from G1 toward S phase and beyond) to yield two daughter cells.
  • Important concept: this cascade demonstrates how extracellular signals control the intracellular transcriptional program via sequential phosphorylation events.

Ras, cancer, and the importance of GTP hydrolysis control

  • Key regulatory molecule: RAS GTPase (GDP-bound inactive vs. GTP-bound active).
  • Normal cycle:
    • RAS–GDP is inactive.
    • SOS activates RAS by promoting GDP-to-GTP exchange → RAS–GTP active.
    • Active RAS propagates signaling leading to cell growth and division.
    • Intrinsic GTPase activity hydrolyzes GTP to GDP, turning RAS back to inactive form; this is the off-switch for the signal.
  • Mutations in RAS (common oncogenic mutations) disrupt hydrolysis, locking RAS in the active GTP-bound state, causing continuous signaling and uncontrolled cell growth.
  • Cancer statistics cited in lecture: about 60% of human tumors harbor RAS mutations across various tissues.
  • Conceptual consequence: when the regulatory hydrolysis step is inhibited or defective, signaling remains on, promoting oncogenesis and tumor formation (in situ tumor formation if unchecked).

Key biophysical and evolutionary context for cellular life

  • Cell membrane and interactions:
    • Membranes are formed by weak, non-covalent interactions (ionic, hydrogen, hydrophobic, van der Waals).
    • Hydrophobic interactions help to form membranes; non-covalent interactions enable dynamic assembly/disassembly.
  • Evolutionary backdrop:
    • Primordial soup and chemical evolution led to the emergence of cellular life.
    • DNA-based replication, transcription, and translation emerged, with viruses and retroviruses (e.g., HIV) interacting with host genomes.
    • Retroviral life cycles involve integration into host chromosomes; inhibition strategies target key steps in viral replication (e.g., certain viral enzymes).
  • Biomarkers and buffers:
    • Biomarkers require buffering systems to sustain life; two primary types of buffers are discussed.
    • Open vs. closed systems are contrasted in context of maintaining homeostasis.
    • Conditions like acidosis and alkalosis are important to understand in terms of cellular and systemic physiology.

Molecular movement, water, and the cellular medium

  • Molecular movement requires a suitable life-sustaining medium, primarily water.
  • Water-related properties to study: polarity, hydrogen bonding, dielectric constant, solvent properties for ions and biomolecules.
  • A strong emphasis on reading and understanding water’s characteristics as central to biochemical processes.

ATP hydrolysis, daily energy needs, and calculation steps

  • ATP hydrolysis energy in vivo vs. in vitro:
    • In vivo, ATP hydrolysis free energy change is commonly around
      $${
      m \, riangle G^{ ext{in vivo}} \