🧪Biophysical Chemistry Unit 5 – Protein Structure and Folding

What's the Big Deal?

• Proteins are essential macromolecules that perform a wide variety of functions in living organisms
• Serve as enzymes catalyzing biochemical reactions, transport molecules, provide structural support, and participate in cell signaling and immune responses
• Understanding protein structure is crucial for deciphering their functions and roles in biological processes
• Protein structure determines how proteins interact with other molecules, such as substrates, ligands, and other proteins
• Misfolding of proteins can lead to various diseases, including Alzheimer's, Parkinson's, and Huntington's disease
  • Accumulation of misfolded proteins can form aggregates that disrupt cellular functions and cause tissue damage
• Studying protein structure and folding is essential for drug design and development
  • Knowing the 3D structure of a protein target allows for rational drug design and optimization of drug-protein interactions

Building Blocks: Amino Acids 101

• Proteins are polymers composed of amino acids linked together by peptide bonds
• There are 20 standard amino acids that serve as the building blocks of proteins
• Amino acids consist of a central carbon atom (Cα) bonded to an amino group (NH2), a carboxyl group (COOH), a hydrogen atom, and a variable side chain (R group)
  • The R group determines the unique properties of each amino acid, such as size, charge, hydrophobicity, and reactivity
• Amino acids can be classified based on the properties of their side chains
  • Nonpolar (hydrophobic) amino acids: Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Proline (Pro), Phenylalanine (Phe), Tryptophan (Trp), Methionine (Met)
  • Polar (hydrophilic) amino acids: Serine (Ser), Threonine (Thr), Cysteine (Cys), Asparagine (Asn), Glutamine (Gln)
  • Charged amino acids: Aspartic acid (Asp), Glutamic acid (Glu), Lysine (Lys), Arginine (Arg), Histidine (His)
• Glycine (Gly) is the simplest amino acid with a hydrogen atom as its side chain, providing flexibility to protein structures
• Amino acids are linked together by peptide bonds formed through a condensation reaction between the carboxyl group of one amino acid and the amino group of another

Primary Structure: The Protein Sequence

• The primary structure of a protein refers to the linear sequence of amino acids linked by peptide bonds
• The sequence of amino acids in a protein is determined by the genetic code, which specifies the order of nucleotides in the corresponding mRNA
• The primary structure is written from the N-terminus (amino end) to the C-terminus (carboxyl end)
• The unique sequence of amino acids in a protein determines its higher-order structures and ultimately its function
• Mutations in the DNA sequence can lead to changes in the primary structure of a protein, potentially altering its function or stability
• Techniques such as Edman degradation and mass spectrometry can be used to determine the primary structure of a protein
  • Edman degradation involves the sequential removal and identification of amino acids from the N-terminus
  • Mass spectrometry measures the mass-to-charge ratio of peptide fragments, allowing for the determination of the amino acid sequence

Secondary Structure: Helices and Sheets

• Secondary structure refers to the local folding patterns of the polypeptide chain, stabilized by hydrogen bonds between the main-chain atoms
• The two main types of secondary structures are α-helices and β-sheets
• α-helices are right-handed spiral conformations stabilized by hydrogen bonds between the carbonyl oxygen of one amino acid and the amino hydrogen of the amino acid four residues ahead (i+4)
  • α-helices have 3.6 amino acids per turn and a pitch of 5.4 Å
  • The side chains of amino acids in an α-helix point outward from the helix axis
• β-sheets are formed by extended polypeptide chains (β-strands) that are arranged side-by-side and stabilized by hydrogen bonds between the main-chain atoms of adjacent strands
  • β-sheets can be parallel (N-termini aligned) or antiparallel (alternating N- and C-termini)
  • The side chains of amino acids in a β-sheet alternate above and below the plane of the sheet
• Other secondary structure elements include turns and loops, which connect α-helices and β-sheets
• The formation of secondary structures is influenced by the amino acid sequence, with certain amino acids having a higher propensity for forming α-helices (Ala, Leu, Met) or β-sheets (Val, Ile, Thr)
• Techniques such as circular dichroism (CD) spectroscopy and Fourier-transform infrared (FTIR) spectroscopy can be used to study the secondary structure composition of proteins

Tertiary Structure: The 3D Puzzle

• Tertiary structure refers to the three-dimensional arrangement of a protein's secondary structure elements and side chains
• The tertiary structure is stabilized by various noncovalent interactions, including hydrogen bonds, van der Waals forces, and hydrophobic interactions
  • Disulfide bonds formed between cysteine residues can also contribute to the stability of the tertiary structure
• The hydrophobic effect plays a crucial role in the folding of proteins, driving the burial of hydrophobic side chains in the protein core and the exposure of hydrophilic side chains on the surface
• The tertiary structure of a protein determines its overall shape, which is essential for its function, such as binding to ligands or catalyzing reactions
• Protein domains are independently folding units within a protein that can have distinct functions
  • Multidomain proteins consist of multiple domains connected by flexible linkers
• Techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM) can be used to determine the tertiary structure of proteins at atomic resolution
  • X-ray crystallography involves the diffraction of X-rays by protein crystals to generate an electron density map
  • NMR spectroscopy measures the magnetic properties of atomic nuclei to determine the distances and angles between atoms in a protein
  • Cryo-EM involves the imaging of frozen protein samples using an electron microscope to generate 3D reconstructions

Quaternary Structure: Protein Teams

• Quaternary structure refers to the arrangement of multiple polypeptide chains (subunits) in a multi-subunit protein complex
• Subunits can be identical (homooligomers) or different (heterooligomers)
• The association of subunits is stabilized by the same noncovalent interactions that stabilize tertiary structure, such as hydrogen bonds, van der Waals forces, and hydrophobic interactions
• Quaternary structure can provide several advantages, including increased stability, regulation of activity, and the formation of binding sites at subunit interfaces
• Examples of proteins with quaternary structure include hemoglobin (tetramer), DNA polymerase (holoenzyme), and ion channels (multi-subunit transmembrane proteins)
• Techniques such as size-exclusion chromatography, analytical ultracentrifugation, and native mass spectrometry can be used to study the quaternary structure of proteins
  • Size-exclusion chromatography separates proteins based on their size and shape, allowing for the determination of the oligomeric state
  • Analytical ultracentrifugation measures the sedimentation velocity and equilibrium of proteins to determine their molecular weight and oligomeric state
  • Native mass spectrometry preserves non-covalent interactions during ionization, enabling the detection of intact protein complexes

Folding Funnel: How Proteins Take Shape

• Protein folding is the process by which a polypeptide chain acquires its native three-dimensional structure
• The folding process is often described using the concept of a folding funnel, which represents the energy landscape of the protein
  • The unfolded state has high conformational entropy and high free energy, while the native state has low conformational entropy and low free energy
• The folding process is driven by the minimization of free energy, which involves a balance between the enthalpic contributions of noncovalent interactions and the entropic cost of reducing conformational freedom
• The folding pathway can involve intermediate states, such as molten globules, which have some secondary structure but lack a well-defined tertiary structure
• Chaperones are proteins that assist in the folding of other proteins by preventing aggregation, facilitating folding, or rescuing misfolded proteins
  • Examples of chaperones include Hsp70, Hsp90, and the GroEL/GroES complex
• Techniques such as fluorescence spectroscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry can be used to study protein folding
  • Fluorescence spectroscopy can monitor the exposure of tryptophan residues during folding
  • Circular dichroism can detect changes in secondary structure content during folding
  • Hydrogen-deuterium exchange mass spectrometry can identify regions of a protein that become protected from exchange during folding

When Things Go Wrong: Misfolding and Diseases

• Protein misfolding occurs when a protein fails to acquire or maintain its native structure, leading to the formation of non-functional or toxic aggregates
• Misfolding can be caused by mutations in the protein sequence, errors in translation, or exposure to environmental stresses such as heat or oxidative stress
• Misfolded proteins can expose hydrophobic regions that are normally buried in the protein core, leading to aggregation through intermolecular interactions
• Protein aggregates can form various structures, such as amorphous aggregates, oligomers, and amyloid fibrils
  • Amyloid fibrils are highly ordered, β-sheet-rich structures that are associated with several neurodegenerative diseases
• Protein misfolding and aggregation are implicated in numerous diseases, including Alzheimer's disease (amyloid-β and tau), Parkinson's disease (α-synuclein), and Huntington's disease (huntingtin)
  • In these diseases, the accumulation of misfolded proteins leads to the formation of toxic aggregates that disrupt cellular functions and cause neuronal death
• Other diseases associated with protein misfolding include cystic fibrosis (CFTR), sickle cell anemia (hemoglobin), and certain types of cancer (p53)
• Strategies for preventing or treating protein misfolding diseases include the use of small molecules that stabilize the native structure, inhibit aggregation, or promote the clearance of misfolded proteins
  • Examples include the use of antibodies against amyloid-β in Alzheimer's disease and the development of pharmacological chaperones for cystic fibrosis