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Protein Structure and Post-Translational Modifications - Vocabulary Flashcards

Energetics of Reversing Processes

  • Key idea: Gibbs free energy, enthalpy, and entropy change when a process is reversed have related sign changes.
  • Relationship between forward and reverse processes:
    • \Delta G{\text{reverse}} = -\Delta G{\text{forward}}
    • \Delta H{\text{reverse}} = -\Delta H{\text{forward}}
    • \Delta S{\text{reverse}} = -\Delta S{\text{forward}}
  • Example discussed in class: if the reverse reaction has a positive Gibbs energy change, an example value given was +38\ \text{kJ/mol} (implying the forward change would be -38\ \text{kJ/mol} if the magnitudes are equal).
  • Conceptual takeaway: the sign of changes and their magnitudes depend on the direction of the process; the magnitude of the energy change is the same in forward and reverse directions, but signs flip.
  • Practical note: the identity of the functional group involved can significantly influence the actual values and signs of enthalpy and entropy changes for the same process in the forward versus reverse direction.

Basic Amino Acid Structure and Designation of the R Group

  • Core structure of an amino acid: amino group, α-carboxyl group, central α-carbon, and a side chain designated as the R group.
  • The R group is the site of chemical variability and largely determines properties like charge, polarity, and hydrophobicity.
  • A generic amino acid includes the backbone and a variable R group (often drawn as R or a label such as "bar" in notes).
  • Isoelectric point concept: the pH at which a molecule has zero net charge (often denoted pI for amino acids; this is the pH where the molecule is neutral overall).
  • Exam question flexibility noted: students could be asked to draw or to show charge states; both approaches are considered acceptable depending on the prompt.

Post-Translational Modifications and Their Effects on Charge and Function

  • After translation, proteins can undergo post-translational modifications (PTMs) that alter structure, charge, stability, and interactions.
  • Phosphorylation
    • Kinases transfer a phosphate group to hydroxyl-containing residues (serine, threonine, tyrosine).
    • Adds negative charge to the modified residue, altering protein interactions and activity.
  • Sulfation (tyrosine sulfation)
    • Addition of a sulfate group to the hydroxyl of tyrosine can introduce a negative charge, depending on the residue and context.
    • Tyrosine’s phenolic OH has a pKa around 10.5, so under physiological conditions (pH ~7) it is typically uncharged unless sulfated.
  • Glycosylation (N- and O-linked)
    • Glycosylation attaches carbohydrate moieties to amino acids, notably asparagine in N-linked glycosylation (Asn-linked) and serine/threonine in O-linked glycosylation.
    • The carbohydrate addition can affect folding, stability, trafficking, and recognition.
  • Acetylation (e.g., acetylated lysine)
    • Acetylation removes a positive charge from the ε-amino group of lysine, which can disrupt interactions that depend on the positive charge, such as DNA binding.
  • Hydroxylation (e.g., hydroxyproline)
    • Addition of hydroxyl groups to proline or other residues can increase polarity and enable new hydrogen bonding, affecting solubility and stability.
  • Methylation, sulfation, and other PTMs discussed illustratively; emphasis on how they shift charge states, hydrogen-bonding patterns, and protein–protein or protein–DNA interactions.
  • Example interpretations from the worksheet discussion:
    • Phosphorylation and sulfation can dramatically shift local and global charge distributions, impacting protein function.
    • Glycosylation introduces bulky carbohydrate groups that can influence folding and extracellular interactions.
    • Acetylation of lysine neutralizes a positive charge, affecting DNA binding and chromatin regulation contexts.
  • Specific examples and notes discussed:
    • Acetyl lysine reduces positive charge and can undermine DNA binding, thus regulating gene expression.
    • Sulfation of tyrosine makes the residue negatively charged, with potential broad impacts depending on the protein context.
    • Asparagine glycosylation adds a carbohydrate moiety; the term “glycosylated asparagine” highlights glycosylation sites.
    • Proline hydroxylation increases polarity in regions where proline was previously less polar.
  • Real-world relevance: PTMs are central to signaling, regulation, and disease; misregulation or aberrant PTMs are linked to various diseases and cellular dysfunctions.

Chirality, D/L System, and Fischer Projections in Amino Acids

  • All standard amino acids are chiral except there is historical student confusion about exceptions; the instructor noted that Lysine has sometimes been described as exceptional in some contexts, but in canonical biochemistry, Lysine is also chiral (has a stereocenter at the α-carbon).
  • Stereochemical representations used in biochemistry:
    • Fischer projection: two-dimensional representation with a cross that implies stereochemistry.
    • In biochemistry, natural amino acids are typically in the L-configuration (NH2 on the left in the standard Fischer projection when the carboxylate is oriented at the top).
  • Determining D vs. L in Fischer projections (with the carboxyl group at the top):
    • Priority order of substituents around the α-carbon is determined by Cahn–Ingold–Prelog rules; the group with higher priority is the amine nitrogen vs hydrogen, etc.
    • Starting from the carboxyl group at the top, determine the order of substituents by priority and assess the rotation (clockwise vs counterclockwise).
    • If the sequence is clockwise, the configuration is D; if counterclockwise, L (with the carboxyl group fixed at the top).
  • Two-dimensional drawings do not imply fixed spatial orientation toward or away from the viewer; the underlying tetrahedral nature of the α-carbon is implicit.
  • Alternative depiction: the R/S system is the absolute configuration used in organic chemistry; biochemistry often uses D/L nomenclature, which corresponds to rotated, simplified conventions in the amino acid context.
  • Practical note: visual tools (e.g., Fischer projections) are used to discuss stereochemistry, but the critical concept is that the stereocenter at the α-carbon gives rise to non-superposable mirror images, hence chirality.
  • Why the orientation of groups matters: the arrangement (left vs right) correlates with the stereochemical configuration and influences how the amino acid fits into enzymes and peptide backbones.

Protein Structure: From Primary to Quaternary

  • Primary structure
    • The covalent sequence of amino acids in a polypeptide.
    • Determined by the coding sequence in DNA, transcribed to RNA, and translated into protein.
    • The order of amino acids defines the potential for higher-order structure and function.
  • Secondary structure
    • Local spatial arrangements stabilized by backbone hydrogen bonds.
    • Major types:
    • Alpha helix: right-handed helix, typically stabilized by N–H···O=C hydrogen bonds between residues i and i+4; approximately 3.6 amino acids per turn.
    • Beta sheets: formed by hydrogen bonds between backbone carbonyls and amide hydrogens across adjacent strands; can be antiparallel or parallel.
    • The R groups (side chains) largely do not participate in the stabilization of the alpha helix backbone, but they influence helix formation and stability via sterics and local interactions.
  • Tertiary structure
    • Overall three-dimensional folding of a single polypeptide chain into a functional domain.
    • Local secondary structure elements (helices, sheets, turns) combine with long-range interactions to produce the compact folded state.
    • The concept that a polypeptide can fold into a specific 3D arrangement where distant residues in sequence can be neighbors in space.
  • Quaternary structure
    • Association of multiple polypeptide chains (subunits) into a functional protein complex.
    • Example in lecture: a four-subunit arrangement forming a quadrilateral assembly, with subunits colored differently to illustrate their spatial arrangement.
  • Protein folding context and diseases
    • Proteins must fold correctly to function; the lab aims to keep proteins in thermodynamically favorable (stable) states under experimental conditions.
    • Misfolding diseases include Alzheimer’s disease (amyloid plaques), Huntington’s disease, Parkinson’s disease, Jakob disease (prion disease), cystic fibrosis (protein misfolding in a transporter), and prion diseases where misfolded proteins propagate misfolding.
  • The enormous diversity of possible sequences
    • For a 100-amino-acid polypeptide, the number of possible sequences is extremely large: 20^{100}
    • This diversity underpins the vast functional repertoire of proteins in biology.
  • Concept: sequence determines structure and function; structure determines function; folding is influenced by the chemical environment (salt, pH, detergents, etc.).
  • The spatial relationship between sequence neighbors and 3D contacts
    • Residues that are close in sequence may be close in space after folding, but residues far apart in sequence can come into proximity in the folded structure.
  • Notion of modular structure and oligomerization
    • Many proteins exist as oligomers; subunits can assemble into quaternary structures with functional implications.
  • Practical takeaway: understanding structure is key to predicting function and designing interventions; software tools help model protein structure (and the lecture pointed toward thinking about real-world software usage).

Alpha Helix: Structure, Stabilization, and Geometry

  • Representations of the alpha helix
    • Various views: ball-and-stick, ribbon, and backbone-plane depictions show how peptide bonds arrange into a helical form.
    • The helix is a compact, right-handed coil (when described as a common form in biology).
  • Geometric characteristics
    • Right-handed: when the helix is rotated so that the backbone winds in a right-handed sense; a helix hand can be inferred using the right-hand rule (thumb up).
    • About 3.6 amino acids per turn; this determines the pitch of the helix and the repeat spacing along the axis.
  • Stabilization mechanism
    • Stabilized primarily by backbone N–H···O=C hydrogen bonds between successive turns, connecting residues i and i+4.
    • The R groups (side chains) largely do not participate in stabilizing the helix backbone itself; their effects arise from steric constraints and specific interactions with the environment.
  • Proline as an exception
    • Proline’s cyclic structure restricts the φ (phi) angle, making it unfavorable in typical alpha helices; proline often disrupts or terminates helices.
  • Ramifications for secondary structure analysis
    • The alpha helix is one of the two canonical secondary structure elements (the other is beta sheets); its formation is dictated by backbone chemistry and allowed dihedral angles, not by a fixed geometry of side chains.

Beta Sheets: Parallel vs Antiparallel and Their Properties

  • Beta sheet geometry
    • Formed by linking strands via backbone hydrogen bonds between C=O and N–H groups of adjacent strands.
    • Strands can run in the same direction (parallel) or in opposite directions (antiparallel).
  • Antiparallel beta sheets
    • Strands run in opposite directions; hydrogen bonds are nearly linear (close to 180°), contributing to strong, highly directional stabilization.
  • Parallel beta sheets
    • Strands run in the same direction; hydrogen bonds are more angled, leading to slightly less optimal alignment and different geometry.
  • Directionality and connectivity
    • Beta sheets can be formed by segments of the same polypeptide or by different polypeptides (interchain interactions).
    • Orientation in structure is determined by the direction of the N→C backbone along each strand; in antiparallel sheets, adjacent strands run opposite directions, while in parallel sheets they run in the same direction.
  • How to determine parallel vs antiparallel (methods discussed in class)
    • Examine backbone directionality (N–C orientation) across strands.
    • Inspect hydrogen bond geometry: linear vs angled H-bonds can indicate antiparallel vs parallel arrangements.
    • Consider whether the strands are near in sequence (in the same polypeptide) or not, which can influence the likelihood of parallel vs antiparallel arrangements in natural proteins.
  • Turns and connections between strands
    • Beta turns and other linking motifs can connect beta strands, contributing to the overall tertiary structure.

Ramachandran Plot and Phi/Psi Dihedral Angles

  • Purpose of Ramachandran analysis
    • Visualizes the allowed regions of backbone dihedral angles: phi (Φ) for the N–Cα bond and psi (Ψ) for the Cα–C bond.
  • Visualization and interpretation
    • The plot shows zones (often colored green/blue) that correspond to commonly observed secondary structures – e.g., right-handed alpha helices, beta sheets, and turns.
    • Regions that fall outside these Allowed/Recommended zones indicate steric clashes or energetically unfavorable conformations.
  • Practical exercise from the worksheet
    • Given sets of (Φ, Ψ) angles, determine which are allowed (green/blue zones) and which are disallowed (dead zones).
    • Example interpretations given in class: angles like (Φ, Ψ) = (−90°, 102°) may be allowed (blue region), while others may fall into disallowed regions.
  • How to determine allowed conformations in practice
    • Look for near-linear backbone hydrogen bonds: N–H···O=C with backbone geometry approaching 180° bond angles.
    • Use the directionality of the polypeptide chain (N→C orientation) to judge whether β-strands in a sheet are parallel or antiparallel.
    • Consider proline’s influence on local phi angles when evaluating possible conformations.
  • Notes on less common regions
    • Left-handed alpha helices and other unusual conformations do appear on Ramachandran plots but are rare for reasonable protein structures.

Turns, Proline, and Beta Turns

  • Beta turns as a class of turns that reverse the direction of the polypeptide chain rapidly.
  • Type I and Type II beta turns (specific turn types discussed):
    • Type II typically requires a proline followed by glycine in the turn region (specific residue pattern for the turn).
    • Type I often involves a proline at a particular position (e.g., position 3) but with a different conformational constraint than Type II.
  • The functional relevance of turns
    • Turns enable sharp reversals in direction, allowing the chain to fold back on itself and connect consecutive secondary structural elements.
  • Structural implications
    • The precise dihedral arrangement around turns is important for the overall topology of proteins and can influence folding pathways and stability.

Protein Folding, Environment, and Disease Implications

  • Folding and stability in vitro
    • Proteins require specific environmental conditions (salt concentration, detergents, pH) to remain properly folded and functional in a test tube.
    • Denaturation or misfolding often correlates with loss of function.
  • Protein folding diseases
    • Alzheimer’s disease is associated with amyloid plaques formed by misfolded proteins.
    • Huntington’s, Parkinson’s diseases are linked to misfolded or aggregated proteins.
    • Jakob disease (transmissible spongiform encephalopathies) and prion diseases involve misfolded proteins that induce misfolding in others.
    • Cystic fibrosis involves misfolding of a transporter protein (CFTR) leading to dysfunction.
  • Structural biology and modeling tools
    • Understanding primary sequence helps predict secondary and tertiary structure.
    • Structural software and modeling approaches are encouraged for exploring protein structure for larger polypeptides (e.g., n ≈ 100 amino acids).

The Big Picture: From Sequence to Function and Beyond

  • Sequence space and biological diversity
    • A polypeptide of length n has an enormous number of possible sequences: 20^{n} possible amino acid sequences.
    • The actual universe of functional proteins arises from selection, folding physics, and cellular context.
  • Relationship between sequence, structure, and function
    • Primary structure (sequence) determines possible secondary structures and tertiary folding.
    • The folded tertiary structure determines the protein’s function; quaternary structure further reorganizes function via subunit interactions.
  • Conceptual takeaways for exams
    • Distinguish between different levels of structure and the forces that stabilize each level (hydrogen bonding, hydrophobic effects, ionic interactions, disulfide bonds).
    • Recognize the impact of PTMs on charge, sterics, and function.
    • Be able to identify and describe differences between alpha helices and beta sheets, including parallel vs antiparallel arrangements and their stabilizing patterns.
    • Understand the role of dihedral angles (phi/psi) in defining secondary structure and how to read a Ramachandran plot.
    • Appreciate how misfolding leads to disease and how oligomeric (quaternary) structures can influence function.

Quick Reference: Key Equations and Facts

  • Gibbs free energy relations for reversals:
    • \Delta G{\text{reverse}} = -\Delta G{\text{forward}}
    • \Delta H{\text{reverse}} = -\Delta H{\text{forward}}
    • \Delta S{\text{reverse}} = -\Delta S{\text{forward}}
  • Isolated energy example: if forward is negative, reverse is positive with equal magnitude (e.g., if forward = -38\ \text{kJ/mol}, reverse = +38\ \text{kJ/mol}).
  • Primary structure concept: sequence of amino acids linked by covalent peptide bonds.
  • Protein length and sequence space: for a polypeptide of length n=100, possible sequences are 20^{100}.
  • Alpha helix specifics: approximately 3.6 residues per turn; stabilization by i to i+4 backbone H-bonds.
  • Beta sheet distinctions: antiparallel vs parallel; linear H-bonds in antiparallel arrangements.
  • Tyrosine pKa and charge considerations: pK_a(y) ≈ 10.5; at pH 7 tyrosine is typically neutral unless modified.
  • Lysine chirality note: in canonical biochemistry, lysine is chiral (the lecture included a claimed exception, which contrasts with standard textbooks).
  • Important PTMs: phosphorylation, sulfation, glycosylation, acetylation, hydroxylation; each can alter charge, hydrogen bonding, and function.