🔬Biophysics Unit 4 – Protein Structure, Folding, and Function

Amino Acids: The Building Blocks

• Amino acids consist of an amino group, a carboxyl group, and a unique side chain (R group) attached to a central carbon atom (α-carbon)
• There are 20 standard amino acids found in proteins, each with a specific R group that determines its chemical properties (polarity, charge, size)
  • Polar amino acids (serine, threonine) are hydrophilic and often found on the surface of proteins
  • Non-polar amino acids (leucine, valine) are hydrophobic and typically buried within the protein core
• Amino acids are joined together by peptide bonds, which form through a condensation reaction between the carboxyl group of one amino acid and the amino group of another
• The sequence of amino acids in a protein is determined by the genetic code, where each codon (triplet of nucleotides) specifies a particular amino acid
• Amino acids can undergo post-translational modifications (phosphorylation, glycosylation) that alter their properties and functions
• The chirality of amino acids is important, as proteins are composed of L-amino acids rather than their mirror-image D-amino acids
• The ionization state of amino acids depends on the pH of the environment, with the isoelectric point (pI) being the pH at which the amino acid has a net charge of zero

Primary Structure: Sequence Matters

• The primary structure of a protein refers to the linear sequence of amino acids connected by peptide bonds
• The amino acid sequence is determined by the genetic code, where each codon in the mRNA corresponds to a specific amino acid
• The N-terminus of a protein is the end with a free amino group, while the C-terminus has a free carboxyl group
• The primary structure of a protein is crucial for determining its higher-order structures (secondary, tertiary, and quaternary) and ultimately its function
• Mutations in the genetic code can lead to changes in the amino acid sequence, potentially altering the protein's structure and function
  • Point mutations involve the substitution of a single amino acid, which may have minor or significant effects depending on the location and nature of the change
  • Insertions and deletions can cause frameshift mutations, drastically altering the amino acid sequence and often resulting in a non-functional protein
• Proteins with similar amino acid sequences often have similar structures and functions, allowing for the prediction of protein function based on sequence homology
• Conserved regions in the amino acid sequence across different species indicate important functional or structural roles for those specific amino acids

Secondary Structure: Helices and Sheets

• Secondary structure refers to the local conformations adopted by the polypeptide chain, primarily 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 another amino acid four residues away
  • The backbone of an α-helix has 3.6 amino acids per turn, with a pitch of 5.4 Å
  • α-helices are often found in globular proteins and are important for protein-protein interactions and DNA binding (leucine zipper motifs)
• β-sheets are formed by extended polypeptide chains that are stabilized by hydrogen bonds between the carbonyl oxygen and amino hydrogen of adjacent strands
  • Parallel β-sheets have strands running in the same direction, while antiparallel β-sheets have strands running in opposite directions
  • β-sheets are often found in the core of globular proteins and are important for protein stability and function (immunoglobulin domains)
• Turns and loops connect α-helices and β-sheets, allowing the polypeptide chain to change direction and form compact globular structures
• The formation of secondary structures is driven by hydrogen bonding, with the amino acid sequence influencing the propensity for certain conformations (proline and glycine are helix breakers)
• Secondary structure prediction algorithms (Chou-Fasman, GOR) use the amino acid sequence to estimate the likelihood of α-helix, β-sheet, or turn formation at each position

Tertiary Structure: The 3D Puzzle

• Tertiary structure refers to the three-dimensional arrangement of a protein's secondary structure elements (α-helices, β-sheets, turns, and loops)
• The tertiary structure is stabilized by various non-covalent interactions, including hydrogen bonds, van der Waals forces, and hydrophobic interactions
  • Salt bridges form between positively and negatively charged amino acid side chains, contributing to protein stability
  • Disulfide bonds between cysteine residues can also stabilize the tertiary structure, particularly in extracellular proteins
• The hydrophobic effect drives the burial of non-polar amino acids in the protein core, while polar and charged residues are often found on the surface
• The tertiary structure of a protein determines its overall shape and function, with specific folds and domains associated with particular functions (catalytic sites, binding pockets)
• Protein folding is a complex process that involves the formation of intermediate states and is guided by the amino acid sequence and the protein's energy landscape
• Misfolded proteins can aggregate and cause cellular dysfunction, leading to diseases such as Alzheimer's and Parkinson's
• Chaperone proteins assist in the proper folding of other proteins, preventing aggregation and ensuring correct tertiary structure formation

Quaternary Structure: Protein Teams

• Quaternary structure refers to the arrangement of multiple polypeptide chains (subunits) to form a functional protein complex
• Subunits can be identical (homooligomers) or different (heterooligomers), with the number of subunits varying from two (dimers) to many (viral capsids)
  • Hemoglobin is a heterotetramer composed of two α and two β subunits, each with an oxygen-binding heme group
  • The ribosome is a large heterooligomeric complex consisting of multiple RNA and protein subunits that work together to synthesize proteins
• Subunit interactions are stabilized by the same non-covalent forces that contribute to tertiary structure stability (hydrogen bonds, van der Waals forces, hydrophobic interactions)
• Quaternary structure can provide several advantages, including increased stability, regulation of activity, and the formation of large, complex structures (cytoskeleton, viral capsids)
• Allosteric regulation often involves quaternary structure changes, where the binding of a ligand to one subunit affects the conformation and activity of other subunits (cooperative oxygen binding in hemoglobin)
• Protein-protein interaction networks are essential for many cellular processes, with quaternary structure playing a key role in the formation and regulation of these networks
• Techniques such as X-ray crystallography, cryo-electron microscopy, and mass spectrometry are used to study the quaternary structure of protein complexes

Protein Folding: Nature's Origami

• Protein folding is the process by which a polypeptide chain acquires its native three-dimensional structure
• The amino acid sequence of a protein contains all the information necessary for it to fold into its native state, known as the thermodynamic hypothesis
• Protein folding is driven by the minimization of free energy, with the native state representing the most stable conformation under physiological conditions
• The hydrophobic effect is a major driving force in protein folding, as non-polar amino acids are buried in the protein core to minimize their contact with water
• Protein folding occurs on a complex energy landscape, with multiple pathways and intermediates leading to the native state
  • The molten globule state is a compact intermediate with native-like secondary structure but lacking well-defined tertiary structure
  • The transition state is the highest energy point along the folding pathway and represents the main barrier to folding
• Chaperone proteins, such as Hsp70 and GroEL/GroES, assist in protein folding by preventing aggregation and providing a favorable environment for folding
• Misfolded proteins can aggregate and form insoluble deposits, leading to diseases such as Alzheimer's (amyloid-β), Parkinson's (α-synuclein), and Huntington's (huntingtin)
• Studying protein folding provides insights into the fundamental principles of protein structure and function, as well as the mechanisms underlying protein misfolding diseases

Factors Affecting Protein Stability

• Protein stability refers to the ability of a protein to maintain its native structure under various environmental conditions
• Temperature influences protein stability, with high temperatures causing denaturation due to the disruption of non-covalent interactions
  • The melting temperature (Tm) is the temperature at which 50% of the protein is denatured
  • Thermophilic proteins from extremophiles have evolved to be stable at high temperatures
• pH affects protein stability by altering the ionization state of amino acid side chains and the formation of salt bridges
  • Proteins are most stable at their isoelectric point (pI), where they have a net charge of zero
  • Extreme pH conditions can cause denaturation by disrupting non-covalent interactions and altering the protein's charge distribution
• Ionic strength influences protein stability by screening electrostatic interactions, with high salt concentrations generally stabilizing proteins
• Chaotropic agents, such as urea and guanidinium chloride, denature proteins by disrupting hydrogen bonds and hydrophobic interactions
• Osmolytes, such as trehalose and glycine betaine, stabilize proteins by preferentially excluding from the protein surface and favoring compact native states
• Mutations can affect protein stability by altering the amino acid sequence and disrupting non-covalent interactions or introducing steric clashes
• Post-translational modifications, such as glycosylation and phosphorylation, can modulate protein stability by altering the protein's surface properties and interactions
• Understanding the factors that influence protein stability is crucial for developing strategies to enhance protein stability for biotechnological and therapeutic applications

Protein Function and Dynamics

• Protein function is intimately linked to its structure, with the three-dimensional arrangement of amino acids determining the protein's specific biological role
• Enzymes are catalytic proteins that accelerate chemical reactions by lowering the activation energy and providing a specific binding site for substrates
  • The active site is the region of an enzyme where the substrate binds and the reaction occurs
  • Enzyme specificity is determined by the complementarity of the active site to the substrate, with key amino acid residues involved in binding and catalysis
• Protein-ligand interactions are essential for many biological processes, such as signal transduction, transport, and regulation
  • Ligand binding often induces conformational changes in the protein, which can modulate its activity or interactions with other molecules
  • The binding affinity and specificity are determined by the complementarity of the ligand to the protein's binding site
• Allostery is a mechanism of regulation where the binding of a ligand at one site affects the activity or binding of another ligand at a distant site
  • Allosteric regulation allows for the fine-tuning of protein activity in response to cellular conditions and signals
  • Conformational changes are often involved in allosteric regulation, with the protein switching between active and inactive states
• Protein dynamics play a crucial role in protein function, with conformational flexibility allowing for substrate binding, catalysis, and regulation
  • Molecular dynamics simulations and NMR spectroscopy are used to study protein dynamics at various time scales
  • Conformational ensembles represent the range of structures a protein can adopt, with the population of each conformation determined by its energy and the environment
• Intrinsically disordered proteins lack a well-defined three-dimensional structure but play important roles in cell signaling and regulation
  • Disordered regions can undergo disorder-to-order transitions upon binding to partners, allowing for specific interactions with multiple targets
  • Disordered proteins are often involved in liquid-liquid phase separation, forming membraneless organelles that compartmentalize cellular processes

Methods for Studying Protein Structure

• X-ray crystallography is a powerful technique for determining the three-dimensional structure of proteins at atomic resolution
  • Protein crystals are grown and exposed to X-rays, producing a diffraction pattern that can be used to calculate the electron density map and build the atomic model
  • X-ray crystallography has been used to solve the structures of many important proteins, including hemoglobin, insulin, and DNA polymerase
• Nuclear magnetic resonance (NMR) spectroscopy is a solution-based method that provides information on protein structure, dynamics, and interactions
  • NMR measures the magnetic properties of atomic nuclei (usually 1H, 13C, and 15N) in a strong magnetic field, yielding chemical shift, coupling constant, and relaxation data
  • NMR can be used to determine the three-dimensional structure of small to medium-sized proteins and to study protein dynamics and ligand binding
• Cryo-electron microscopy (cryo-EM) is a technique for studying the structure of large protein complexes and macromolecular assemblies
  • Samples are rapidly frozen in a thin layer of vitreous ice and imaged using an electron microscope, preserving their native structure
  • Single-particle analysis and tomography are used to reconstruct three-dimensional models from the two-dimensional images
• Circular dichroism (CD) spectroscopy is a technique for studying the secondary structure composition of proteins in solution
  • CD measures the differential absorption of left- and right-circularly polarized light by chiral molecules, such as proteins
  • The CD spectrum provides information on the relative amounts of α-helices, β-sheets, and random coils in a protein sample
• Fluorescence spectroscopy is used to study protein folding, dynamics, and interactions
  • Intrinsic fluorescence from tryptophan residues or extrinsic fluorescent labels can be used to monitor conformational changes and binding events
  • Förster resonance energy transfer (FRET) measures the distance between two fluorophores, providing information on protein conformation and interactions
• Mass spectrometry is a technique for studying protein mass, composition, and interactions
  • Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are used to ionize proteins for mass spectrometric analysis
  • Mass spectrometry can be used to identify proteins, characterize post-translational modifications, and study protein-protein and protein-ligand interactions

Protein Structure-Function Relationships

• The structure of a protein determines its function, with the three-dimensional arrangement of amino acids creating specific binding sites, catalytic centers, and interaction surfaces
• Protein domains are conserved structural and functional units that can be found in multiple proteins
  • Domains often fold independently and can be combined in different ways to create proteins with diverse functions
  • Examples of common domains include the kinase domain, the DNA-binding domain, and the SH2 domain
• Active sites in enzymes are composed of specific amino acid residues that are positioned to bind the substrate and catalyze the reaction
  • The catalytic triad in serine proteases (Ser-His-Asp) is an example of a conserved active site motif that is essential for the hydrolysis of peptide bonds
  • Mutations in active site residues can drastically alter enzyme activity and specificity
• Protein-protein interaction interfaces are formed by complementary surfaces on the interacting proteins
  • Hydrophobic, electrostatic, and hydrogen-bonding interactions contribute to the specificity and stability of protein-protein interfaces
  • Hotspot residues are critical amino acids that contribute disproportionately to the binding energy of the interaction
• Protein-ligand interactions are mediated by specific binding pockets that are complementary to the ligand in terms of shape, size, and chemical properties
  • The binding of oxygen to hemoglobin is an example of a protein-ligand interaction that is essential for the transport of oxygen in the blood
  • Ligand binding can induce conformational changes in the protein, which can modulate its activity or interactions with other molecules
• Protein misfolding and aggregation can lead to loss of function and the formation of toxic species
  • Amyloid fibrils are ordered aggregates of misfolded proteins that are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's
  • Strategies to prevent or reverse protein misfolding, such as the use of small molecule chaperones or antibodies, are being explored as potential therapies for these diseases
• Rational drug design involves the use of protein structure information to guide the development of new therapeutic agents
  • Structure-based drug design uses the three-dimensional structure of the target protein to identify and optimize small molecule ligands that bind specifically and modulate its activity
  • Examples of drugs developed using structure-based design include HIV protease inhibitors and kinase inhibitors used in cancer therapy