👩‍🔬Intro to Biotechnology Unit 6 – Protein Structure and Engineering

Basics of Protein Structure

• Proteins are essential macromolecules that play crucial roles in virtually all biological processes within living organisms
• Composed of one or more polypeptide chains, which are linear sequences of amino acids linked together by peptide bonds
• The specific sequence of amino acids in a protein, known as its primary structure, determines its unique three-dimensional shape and function
• Proteins can be classified based on their overall shape and function, such as globular proteins (enzymes, antibodies) and fibrous proteins (collagen, keratin)
• The structure of a protein is maintained by various non-covalent interactions, including hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions
• Proteins are synthesized by ribosomes in the cell, following the genetic instructions encoded in the DNA and transcribed into mRNA
• After synthesis, proteins may undergo post-translational modifications (glycosylation, phosphorylation) that can alter their structure and function

Amino Acids and Peptide Bonds

• Amino acids are the building blocks of proteins, consisting of an amino group ($-NH_2$), a carboxyl group ($-COOH$), and a variable side chain ($R$) attached to a central carbon atom ($C_\alpha$)
• There are 20 standard amino acids found in proteins, each with a unique side chain that determines its chemical properties (polarity, charge, size)
• Amino acids are linked together by peptide bonds, which are formed through a condensation reaction between the carboxyl group of one amino acid and the amino group of another, releasing a water molecule
• The formation of peptide bonds creates a repeating backbone structure in the protein, consisting of the amino nitrogen, the $C_\alpha$, and the carbonyl carbon
• The sequence of amino acids in a protein is determined by the genetic code, where each three-nucleotide codon in the mRNA specifies a particular amino acid
• The peptide bond has a partial double bond character due to resonance, which restricts rotation around the bond and contributes to the planar structure of the protein backbone
• The peptide bond has a trans configuration in most cases, meaning that the $C_\alpha$ atoms of adjacent amino acids are on opposite sides of the peptide bond plane

Levels of Protein Organization

• Protein structure is organized into four distinct levels: primary, secondary, tertiary, and quaternary structure
• Primary structure refers to the linear sequence of amino acids in a polypeptide chain, which is determined by the genetic code and forms the basis for higher levels of organization
• Secondary structure describes the local folding patterns of the polypeptide chain, primarily $\alpha$-helices and $\beta$-sheets, which are stabilized by hydrogen bonds between the backbone atoms
  • $\alpha$-helices are right-handed spiral conformations with 3.6 amino acids per turn, stabilized by hydrogen bonds between the carbonyl oxygen of one amino acid and the amino hydrogen of the amino acid four residues ahead in the sequence
  • $\beta$-sheets are extended conformations with the polypeptide chain forming a zigzag pattern, stabilized by hydrogen bonds between backbone atoms of adjacent strands, which can be parallel or antiparallel
• Tertiary structure refers to the overall three-dimensional arrangement of a single polypeptide chain, resulting from the packing of secondary structure elements and the interactions between side chains
• Quaternary structure describes the assembly of multiple polypeptide chains (subunits) into a functional protein complex, stabilized by non-covalent interactions between the subunits (hemoglobin, DNA polymerase)
• The different levels of protein organization work together to create a unique three-dimensional structure that enables the protein to perform its specific biological function

Protein Folding and Stability

• Protein folding is the process by which a polypeptide chain acquires its native three-dimensional structure, which is essential for its biological function
• The folding process is driven by the minimization of the protein's free energy, as the polypeptide chain explores different conformations to reach its most stable state
• The native state of a protein is determined by its amino acid sequence, as the interactions between side chains and the surrounding environment guide the folding process
• Protein folding is influenced by various factors, including the properties of the amino acid side chains (hydrophobicity, charge), the cellular environment (pH, temperature, ionic strength), and the presence of chaperone proteins that assist in the folding process
• Misfolded proteins can lead to aggregation and the formation of insoluble deposits, which are associated with various diseases (Alzheimer's, Parkinson's, Huntington's)
• Protein stability is maintained by a balance of non-covalent interactions, including hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions
  • Hydrogen bonds form between the backbone atoms and side chains of polar amino acids, contributing to the stability of secondary structures and the overall folding of the protein
  • Ionic bonds (salt bridges) form between positively and negatively charged side chains, stabilizing the tertiary structure of the protein
  • Van der Waals forces are weak attractive forces between atoms that arise from temporary dipoles, contributing to the close packing of the protein interior
  • Hydrophobic interactions drive the burial of non-polar side chains in the protein core, away from the aqueous environment, which is a major determinant of protein stability
• Proteins can be denatured by various factors that disrupt their native structure, such as high temperature, extreme pH, and chemical denaturants (urea, guanidinium chloride), leading to a loss of biological function

Protein Function and Interactions

• Proteins perform a wide range of functions in living organisms, including catalysis (enzymes), transport (hemoglobin), structural support (collagen), signaling (hormones), and defense (antibodies)
• The specific function of a protein is determined by its unique three-dimensional structure, which creates binding sites and active sites that interact with other molecules
• Enzymes are proteins that catalyze chemical reactions by lowering the activation energy and stabilizing transition states, enabling reactions to occur at much faster rates under physiological conditions
  • Enzyme activity is regulated by various mechanisms, such as allosteric regulation (binding of effectors), covalent modification (phosphorylation), and changes in pH or temperature
  • Enzymes exhibit substrate specificity, binding only to specific molecules that fit into their active site, which is determined by the arrangement of amino acid side chains
• Proteins can interact with other molecules, such as ligands (substrates, inhibitors), other proteins, and nucleic acids (DNA, RNA), through specific binding sites on their surface
• Protein-protein interactions are essential for many biological processes, such as signal transduction (receptor-ligand complexes), enzyme regulation (inhibitors, activators), and the formation of multi-protein complexes (ribosomes, proteasomes)
  • Protein-protein interactions are mediated by complementary surface features, such as shape complementarity, electrostatic interactions, and hydrophobic contacts
  • Specific protein-protein interaction domains, such as SH2, SH3, and PDZ domains, recognize and bind to specific sequence motifs on their partner proteins
• Proteins can also interact with nucleic acids, such as transcription factors that bind to specific DNA sequences to regulate gene expression, and RNA-binding proteins that are involved in RNA processing and translation
• Understanding protein function and interactions is crucial for designing drugs that can modulate protein activity, either by inhibiting or activating specific targets involved in disease processes

Techniques for Studying Protein Structure

• Various experimental techniques are used to determine the three-dimensional structure of proteins, providing insights into their function and interactions
• X-ray crystallography is a powerful method for determining the atomic-level structure of proteins, by analyzing the diffraction patterns of X-rays scattered by a protein crystal
  • Protein crystals are grown from purified protein solutions under specific conditions (pH, salt concentration, precipitants) that promote the ordered packing of molecules
  • The diffraction data is used to calculate an electron density map, which is then interpreted to build a model of the protein structure
  • X-ray crystallography has been used to determine the structures of many important proteins, such as hemoglobin, insulin, and DNA polymerase
• Nuclear magnetic resonance (NMR) spectroscopy is another technique for determining protein structure in solution, by measuring the magnetic properties of atomic nuclei ($^1H$, $^13C$, $^15N$) in the protein
  • NMR provides information about the local environment of each nucleus, which can be used to determine the distances and angles between atoms and to calculate the overall protein structure
  • NMR is particularly useful for studying protein dynamics and interactions, as it can provide information about the flexibility and conformational changes of proteins in solution
• Cryo-electron microscopy (cryo-EM) is an emerging technique for determining the structure of large protein complexes and membrane proteins, by imaging frozen protein samples with an electron microscope
  • Protein samples are rapidly frozen in a thin layer of vitreous ice, preserving their native structure and orientation
  • The electron micrographs are processed to generate a three-dimensional reconstruction of the protein structure, which can reach near-atomic resolution in some cases
• Other techniques for studying protein structure and interactions include:
  • Circular dichroism (CD) spectroscopy, which measures the differential absorption of left- and right-handed circularly polarized light by proteins, providing information about their secondary structure content
  • Fluorescence spectroscopy, which uses the intrinsic fluorescence of aromatic amino acids (tryptophan, tyrosine) or extrinsic fluorescent labels to study protein folding, dynamics, and interactions
  • Surface plasmon resonance (SPR), which measures the binding kinetics and affinity of protein-ligand interactions by detecting changes in the refractive index at a sensor surface
  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS), which probes the solvent accessibility and dynamics of protein regions by measuring the exchange of hydrogen atoms with deuterium in the solvent

Protein Engineering Methods

• Protein engineering involves the rational design and modification of proteins to alter their structure, function, or stability for specific applications
• Site-directed mutagenesis is a technique for introducing specific amino acid substitutions into a protein sequence, by modifying the DNA sequence that encodes the protein
  • Oligonucleotide primers containing the desired mutation are used to amplify the modified DNA sequence, which is then expressed in a suitable host organism (bacteria, yeast, mammalian cells)
  • Site-directed mutagenesis can be used to study the role of specific amino acids in protein function, to improve enzyme activity or specificity, or to introduce new functional groups for bioconjugation
• Directed evolution is a powerful approach for generating proteins with improved or novel functions, by mimicking the process of natural selection in the laboratory
  • Large libraries of protein variants are created by random mutagenesis (error-prone PCR) or DNA shuffling (recombination of gene fragments), and screened for desired properties (activity, stability, binding affinity)
  • The best-performing variants are selected and subjected to additional rounds of mutagenesis and screening, leading to the progressive accumulation of beneficial mutations
  • Directed evolution has been used to engineer enzymes with enhanced catalytic efficiency, altered substrate specificity, and improved stability under extreme conditions (temperature, pH, solvents)
• Rational protein design involves the use of computational methods to predict and design protein structures with desired functions, based on the principles of protein folding and the knowledge of existing protein structures
  • Protein design algorithms search for amino acid sequences that are compatible with a given backbone structure, optimizing the interactions between side chains and the surrounding environment
  • Designed proteins can be synthesized and experimentally characterized to validate the computational predictions and to optimize their properties
  • Rational protein design has been used to create novel enzymes, protein-based materials, and therapeutic proteins with improved stability and binding affinity
• Protein conjugation and fusion techniques allow the covalent attachment of functional molecules (fluorescent labels, drugs, polymers) or the genetic fusion of protein domains to create chimeric proteins with new properties
  • Bioconjugation methods exploit the reactivity of specific amino acid side chains (lysine, cysteine) or introduce non-natural amino acids with unique functional groups for site-specific labeling
  • Protein fusion involves the genetic linking of two or more protein domains, such as the fusion of an antibody fragment to an enzyme or a targeting peptide, to create bifunctional proteins with enhanced specificity and activity
• Structural biology and bioinformatics tools are essential for guiding protein engineering efforts, by providing insights into the structure-function relationships of proteins and enabling the rational design of modifications
  • Protein structure databases (PDB) and sequence databases (UniProt) are valuable resources for identifying homologous proteins, conserved motifs, and functionally important residues
  • Molecular modeling and simulation techniques, such as homology modeling, molecular dynamics, and docking, can predict the effects of mutations on protein structure and interactions, and guide the design of novel proteins

Applications in Biotechnology

• Protein engineering has numerous applications in biotechnology, ranging from the production of industrial enzymes and biopharmaceuticals to the development of new materials and diagnostic tools
• Engineered enzymes are widely used in various industrial processes, such as food processing, textile manufacturing, and biofuel production, due to their high specificity, efficiency, and sustainability
  • Examples include the use of engineered cellulases and hemicellulases for the degradation of plant biomass into fermentable sugars for bioethanol production, and the use of engineered proteases and lipases in detergents for improved cleaning performance
  • Engineered enzymes can also be used for the synthesis of fine chemicals, such as chiral compounds and pharmaceuticals, by exploiting their ability to catalyze stereospecific reactions under mild conditions
• Protein therapeutics, such as monoclonal antibodies, hormones, and growth factors, are a major class of biopharmaceuticals that benefit from protein engineering approaches
  • Antibody engineering techniques, such as humanization and affinity maturation, are used to improve the efficacy, safety, and pharmacokinetics of therapeutic antibodies for the treatment of cancer, autoimmune diseases, and infectious diseases
  • Engineered protein hormones, such as insulin analogs and growth hormone variants, are designed to have improved stability, solubility, and bioavailability compared to their native counterparts
  • Fusion proteins, such as immunocytokines and antibody-drug conjugates, combine the targeting specificity of antibodies with the therapeutic activity of cytokines or small-molecule drugs, enabling the selective delivery of therapeutics to disease sites
• Protein-based biomaterials are engineered to have specific mechanical, optical, or biological properties for applications in tissue engineering, drug delivery, and biosensing
  • Engineered protein scaffolds, such as elastin-like polypeptides and silk-like proteins, can be designed to self-assemble into nanofibers, hydrogels, or films with tunable properties, such as elasticity, biodegradability, and cell adhesion
  • Protein-based nanoparticles, such as virus-like particles and protein cages, can be engineered to encapsulate and deliver drugs, genes, or imaging agents to specific cells or tissues, by displaying targeting ligands on their surface
  • Protein-based biosensors and diagnostic tools exploit the specific binding and recognition properties of engineered proteins, such as antibody fragments and aptamers, to detect biomarkers, pathogens, or environmental pollutants with high sensitivity and selectivity
• Protein engineering is also applied in the field of synthetic biology, which aims to create novel biological systems and organisms with desired functions
  • Engineered protein circuits, such as transcriptional regulators and signaling pathways, can be designed to control gene expression and cellular behavior in response to specific stimuli, enabling the programming of complex biological functions
  • Engineered metabolic pathways, consisting of multiple enzymes and regulatory proteins, can be optimized to produce high-value compounds, such as biofuels, pharmaceuticals, and biomaterials, from renewable feedstocks or waste streams
  • Engineered protein machines, such as molecular motors and nanopores, can be designed to perform specific tasks, such as controlled transport, sensing, or actuation, at the nanoscale, with potential applications in drug delivery, biosensing, and nanorobotics