🔮Chemical Basis of Bioengineering I Unit 9 – Protein Structure and Function Analysis

Key Concepts

• Proteins perform a vast array of functions within organisms, including catalyzing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another
• Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes and ultimately results in protein folding into a specific three-dimensional structure that determines its activity
• The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code
• The genetic code specifies 20 standard amino acids, but in certain organisms, the genetic code can include selenocysteine and pyrrolysine
• Short amino acid sequences within proteins often act as recognition sites for other proteins and small molecules, and are known as epitopes

Protein Basics

• Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues
• Proteins can be informally referred to as the workhorses of the cell because they play critical roles in essentially all biological processes
• Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism
• Proteins also have structural or mechanical functions (actin and myosin in muscle and the proteins in the cytoskeleton)
• Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle
• Proteins can also function as sources of amino acids for organisms that do not synthesize all amino acids they need (essential amino acids)
• Proteins can be classified based on their physical properties (solubility, isoelectric point, and molecular weight), chemical composition, and biological function

Amino Acids and Peptide Bonds

• Amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid
• The key elements of an amino acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other elements are found in the side chains of certain amino acids (sulfur, selenium, phosphorus)
• About 500 naturally occurring amino acids are known, though only 20 appear in the genetic code
• Amino acids can be classified according to the properties of their side chains (acidic, basic, polar, nonpolar, aromatic, aliphatic)
• Peptide bonds are formed by a biochemical reaction that extracts a water molecule as it joins the amino group of one amino acid to the carboxyl group of a neighboring amino acid
  • Peptide bonds have two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so the alpha carbons are roughly coplanar
  • The peptide bond has a partial double bond character, which restricts the rotation around the bond (dihedral angles $\phi$ and $\psi$) and keeps the peptide group planar
• Polypeptides are linear chains of amino acids connected by peptide bonds between the carboxyl group of one amino acid and the amino group of the next

Levels of Protein Structure

• The primary structure of a protein refers to the linear sequence of amino acids in the polypeptide chain
  • The primary structure is held together by covalent bonds (peptide bonds) made during the process of protein biosynthesis
  • The two ends of the polypeptide chain are referred to as the N-terminal end (amino-terminal end) and the C-terminal end (carboxyl-terminal end)
• Secondary structure refers to highly regular local sub-structures stabilized by hydrogen bonds, the most common examples being alpha helices and beta sheets
  • The alpha helix ($\alpha$-helix) is a right-handed coiled or spiral conformation in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid located three or four residues earlier along the protein sequence
  • Beta sheets ($\beta$-sheets) are formed by backbone hydrogen bonds between individual beta strands, which may be oriented in a parallel or antiparallel configuration
  • Turns and loops are other elements of secondary structure that connect $\alpha$-helices and $\beta$-sheets
• Tertiary structure refers to the three-dimensional structure of monomeric and multimeric protein molecules
  • Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and post-translational modifications
• Quaternary structure exists in proteins consisting of two or more identical or different polypeptide chains (subunits) that operate as a single functional unit (multimers)
  • The subunits are held together by the same non-covalent interactions and disulfide bonds as the tertiary structure
  • Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers (dimers, trimers, tetramers, etc. based on the number of subunits)

Protein Folding and Stability

• Protein folding is the physical process by which a protein chain acquires its native three-dimensional structure, a conformation that is usually biologically functional
• Protein folding occurs in a cellular compartment called the endoplasmic reticulum for secreted proteins and in the cytoplasm for all other proteins
• Protein folding is driven by the search to find the most thermodynamically stable conformation (the native state) among all possible conformations
• The folding process is a spontaneous process that is mainly guided by hydrophobic interactions, formation of intramolecular hydrogen bonds, and van der Waals forces
  • Hydrophobic residues tend to cluster together in the interior of the molecule (hydrophobic collapse), whereas hydrophilic residues are more likely to be in contact with the aqueous environment and the solvent
• Chaperone proteins assist the non-covalent folding of proteins and the formation of the proper tertiary structure
• Protein stability refers to the tendency of a protein to maintain its native state conformation
  • Protein stability is governed by the collective strength of the non-covalent interactions of the native state compared to the unfolded state
  • Factors affecting protein stability include pH, temperature, ionic strength, and the presence of denaturants, organic solvents, or detergents

Protein Function and Domains

• The function of a protein is directly dependent on its three-dimensional structure
• Enzymes, the most common type of protein, act as catalysts to lower the activation energy of biochemical reactions
  • The catalytic activity of enzymes involves the binding of their substrates and the subsequent reaction of those substrates
  • Enzymes are usually highly specific, only acting on substrates with particular stereochemical configurations
• Many proteins are involved in the process of cell signaling and signal transduction
  • Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues
  • Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell
• Membrane transport proteins are responsible for the movement of ions and small molecules across cell membranes
  • These include ion channels, proton pumps, and g protein-coupled receptors
• Structural proteins confer stiffness and rigidity to otherwise fluid biological components
  • The cytoskeleton, an extensive network of protein filaments, maintains the shape of a cell
  • Actin and tubulin are globular proteins that polymerize to form long, stiff fibers that comprise the cytoskeleton
• Protein domains are distinct functional and/or structural units in a protein that are usually evolutionarily conserved
  • Domains usually contain between 40 and 350 amino acids and are frequently observed as recurring sequence or structural units
  • Proteins are often composed of several structural domains, each of which can have a specific function (binding activity, catalytic activity, etc.)

Analytical Techniques

• Protein purification involves a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues, or whole organisms
  • Protein purification is vital for the characterization of the function, structure, and interactions of the protein of interest
  • The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins
  • Separation steps exploit differences in protein size, physico-chemical properties, binding affinity, and biological activity
• Protein sequencing encompasses techniques to determine the amino acid sequence of a protein
  • Edman degradation is a method of sequencing amino acids in a peptide, one at a time from the N-terminal end
  • Mass spectrometry methods can be used to determine the masses and sequences of proteins and peptides
• Protein structure determination techniques aim to determine the three-dimensional structure of proteins
  • X-ray crystallography is a technique that exploits the fact that X-rays are diffracted by crystals to determine the arrangement of atoms within a crystal from the angles and intensities of these diffracted beams
  • Nuclear magnetic resonance (NMR) spectroscopy exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained
  • Cryo-electron microscopy (cryo-EM) is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures in vitreous ice
• Protein-protein interactions can be studied using a variety of techniques
  • Yeast two-hybrid screening is a molecular biology technique used to discover protein-protein interactions by testing for physical interactions between two proteins
  • Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand
  • Surface plasmon resonance is a technique to study protein-protein interactions by detecting molecular interactions at the surface of a sensor chip

Applications in Bioengineering

• Protein engineering involves the design and construction of novel proteins, usually by manipulation of their amino acid sequences
  • Rational design uses detailed knowledge of the structure and function of the protein to make desired changes, such as increasing its stability or altering its specificity
  • Directed evolution is a method used in protein engineering that mimics the process of natural selection to evolve proteins toward a user-defined goal
• Recombinant proteins are proteins produced by recombinant DNA technology in which the gene coding for the protein of interest is cloned into an expression vector, which is then introduced into a host organism for expression
  • Recombinant proteins are used in the production of therapeutic proteins (insulin, growth hormone), industrial enzymes, and research reagents
• Protein-based biomaterials are materials derived from proteins for potential biomedical applications
  • Collagen, the most abundant protein in mammals, is used in cosmetic surgery and wound healing applications
  • Spider silk proteins are being investigated for use in bulletproof vests and medical sutures due to their high tensile strength and elasticity
• Protein-based sensors and devices exploit the specificity and sensitivity of proteins for the detection of target molecules
  • Antibodies are used in immunoassays (ELISA, Western blot) for the specific detection of proteins
  • Enzymes are used in biosensors for the detection of specific analytes (glucose oxidase for glucose sensing)
• Protein-based drugs and therapeutics use proteins as the active pharmaceutical ingredient for the treatment of diseases
  • Monoclonal antibodies (Humira, Herceptin) are used for the treatment of autoimmune diseases and cancer
  • Enzyme replacement therapy involves the administration of a functional enzyme to replace a deficient or absent enzyme in patients with genetic disorders (Gaucher's disease, Fabry disease)