2- BCC 3D Protein Structure Handouts.pdf

Chapter 4: Proteins Three Dimensional Structure and Protein Folding Nükhet Aykın-Burns, PhD Division of Radiation Health...

Chapter 4: Proteins Three Dimensional Structure and Protein Folding Nükhet Aykın-Burns, PhD Division of Radiation Health Pharmaceutical Sciences Biomed II, Room 441A-2 LEARNING OBJECTIVES: At the end of this lecture, students should be able to: 1. Understand the main roles of proteins. 2. Describe the building blocks, bonds and specific interactions involved in protein structure. 3. Describe the primary, secondary, tertiary, quaternary structures of proteins in detail. 4. Describe the types of bonding interactions between "side chains” are involved in protein folding 5. Explain the mechanisms of protein folding and denaturation 6. Name and describe the diseases associated with protein misfolding. 7. Explain the role and function of chaperones.  Four Levels of Protein Structure. § Unlike most organic polymers, protein molecules adopt a specific three- dimensional conformation. § This structure is able to fulfill a specific biological function. § This structure is called the native fold. § The native fold has a large number of favorable interactions within the protein. § Levels of structure in proteins. The primary structure consists of a sequence of amino acids linked together by peptide bonds and includes any disulfide bonds. The resulting polypeptide can be arranged into units of secondary structure, such as an helix. The helix is a part of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make up the quaternary structure of the multisubunit protein.  Favorable Interactions in Proteins Hydrophobic effect The release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy. Ionic bonds The bonds formed via electrostatic attraction between oppositely charged ions. Hydrogen bonds Interaction of N−H and C=O of the peptide bond leads to local regular structures such as α-helices and β-sheets. They can also form within α-helices between β-sheets. Disulfide bonds The covalent bonds that are derived from two thiol (-SH) groups. London dispersion Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein. Electrostatic interactions Long-range strong interactions between permanently charged groups (e.g. H-bonds) Salt bridges, especially those buried in the hydrophobic environment, strongly stabilize the protein.  Primary Structure: The Peptide Bond Amide Bond 1 kDa = 1000 Da Ser-Gly-Tyr-Ala-Leu (SGYAL) § A peptide bond (also called amide bond) is formed by linking the α-carboxyl group of one amino acid to α-amino group of another amino acid via the release of a H2O molecule, thus it is a (dehydration) condensation reaction. § Polypeptides consist of amino acids linked by a peptide bond. § The polypeptide chain consists of a repeating part called the main chain or backbone and a variable part consisting of the distinctive amino acid side chains. § Proteins – most natural polypeptide chains contain between 50 and 2000 amino acid residues. § A polypeptide chain has directionality. The amino terminal end is taken as the beginning of the polypeptide chain. The carboxyl terminal end is the end of the polypeptide chain. The primary structure is always written from the amino terminal to the carboxyl terminal or left to right. § The backbone has hydrogen-bonding potential because of the carbonyl groups and hydrogen atoms that are bonded to the nitrogen of the amine group. § Synthesis of protein primary structure require input of free energy. Peptide bonds are kinetically stable, lifetime of 1000 yrs in the absence of catalyst. § The mass of protein – expressed in units of dalton, one dalton is equal to one atomic mass unit.  Primary Structure: The Peptide Bond Rigid Partial double bond Nearly planar No rotation Favors trans configuration with a large dipole moment Protein folding is determined by a network of interactions between amino acids in the polypeptide, thus the final structure of the protein chain is determined by its amino acid sequence. § The structure of the protein is partially dictated by the properties of the peptide bond. § Each peptide bond has some double-bond character due to resonance and cannot rotate. § The peptide bond is a resonance hybrid of two canonical structures. § The resonance causes the peptide bonds: to be less reactive compared with esters to be quite rigid and nearly planar to exhibit a large dipole moment in the favored trans configuration  Secondary Structure: α-helix and β-sheet The organization around the peptide bond, paired with the identity of the R groups, determines the secondary structure of the protein. Pauling, Corey and Branson PNAS, 1951 § Secondary structure refers to a local spatial arrangement of the polypeptide backbone formed by hydrogen bonds between peptide NH and CO groups of amino acids that are near one another in the primary structure. § The α helix, β sheets/strands and turns are prominent examples of secondary structure. § Other regions of the polypeptide chain form non-regular, non-repetitive secondary structures such as loops and coils. § In April 1951 Pauling, Corey and Branson published “The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain,” in the Proceedings of the National Academy of Sciences. § The elucidation of the structure of the α-helix is a landmark in biochemistry because it demonstrated that the conformation of a polypeptide chain can be predicted if the properties of its components are rigorously and precisely known.  Secondary Structure: α-helix Macroscopic dipole moment ü All of the backbone CO and NH groups form hydrogen bonds except those at the end of the helix. § The α helix is a tightly coiled rod like structure, with the R groups bristling out from the axis of the helix. § The CO group of each amino acid forms a hydrogen bond with the NH group of the amino acid that is situated four residues ahead in the sequence. All of the backbone CO and NH groups form hydrogen bonds except those at the end of the helix. § Amino acids spaced three and four apart in the sequence are spatially quite close to one another in an α-helix. § Recall that the peptide bond has a strong dipole moment. C−O (carbonyl) negative N−H (amide) positive § All peptide bonds in the α helix have a similar orientation. § The α helix has a large macroscopic dipole moment that is enhanced by unpaired amides and carbonyls near the ends of the helix.  Secondary Structure: α-helix structure and stability Strong helix formers Ferritin Not all polypeptide sequences adopt α-helical structures. Collagen Helix Breakers § Essentially all α helices found in proteins are right-handed. § Right-handed helices are energetically more favorable because there is less steric clash between the side chains and the backbone. § A largely α-helical protein -> Ferritin, an iron-storage protein, is built from a bundle of α helices. § Not all polypeptide sequences adopt α-helical structures. § Small hydrophobic residues such as Ala and Leu are strong helix formers. § Pro acts as a helix breaker because the rotation around the N-Ca (φ-angle) bond is impossible. § Gly acts as a helix breaker because the tiny R group supports other conformations. § Attractive or repulsive interactions between side chains 3 to 4 amino acids apart will affect formation.  Secondary Structure: β-strand and β-sheet Conformationally less stable, so rare Both α helices and β sheets allow the maximum number of H-bonds within the interior of a polypeptide § Structure of a β-Strand. The side chains are alternately above and below the plane of the strand. § A polypeptide chain, called a β strand is almost fully extended rather than being tightly coiled as in the α helix. § Beta sheet are stabilized by hydrogen bonding between polypeptide strands (inter- strand). § The side chains of adjacent amino acids point in opposite directions. § β strands are rare because they are conformationaly less stable. However, when two adjacent β strands line up they can form hydrogen bonds. This creates a β sheet. § Adjacent chains in a β sheet can run in opposite directions (antiparallel β sheet) or in the same direction (parallel β sheet) or mixed. § Both α helices and β sheets allow the maximum number of H-bonds within the interior of a polypeptide. § Parallel sheets are less stable than antiparallel sheet, possibly because the hydrogen bonds are distorted. § In parallel β sheets, the H-bonded strands run in the same direction. Hydrogen bonds between strands are bent (weaker). § In antiparallel β sheets, the H-bonded strands run in opposite directions. Hydrogen bonds between strands are linear (stronger) § β sheets in globular proteins have a right handed curl or twist when viewed along the polypeptide backbone.  Secondary Structure: β-turns and β-loops ü Turns are stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence. ü Turns and loops invariably lie on the surfaces of proteins and thus often participate in interactions between proteins and the environment. § β turns occur frequently whenever strands in b sheets change the direction. § The 180° turn is accomplished over four amino acids. § The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence. § They are often found on the surface of the proteins and facilitate formation of a compact globular shape. § Proline in position 2 or glycine in position 3 are common in b turns.  Tertiary Structure: overall spatial arrangement of atoms in a protein ü A system is more thermodynamically stable when hydrophobic groups are clustered rather than extended into the aqueous surroundings. § Protein tertiary structure - a protein's geometric shape. It refers to the overall spatial arrangement of atoms in a protein. § The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. § This final shape is determined by a variety of bonding interactions between the "side chains" on the amino acids. § These bonding interactions may be stronger than the hydrogen bonds between amide groups holding the helical structure. § As a result, bonding interactions between "side chains" may cause a number of folds, bends, and loops in the protein chain. Different fragments of the same chain may become bonded together. § Water-soluble proteins fold into compact structures within nonpolar cores § In an aqueous environment, protein folding is driven by the strong tendency of hydrophobic residue to be excluded from water. § A system is more thermodynamically stable when hydrophobic groups are clustered rather than extended into the aqueous surroundings. § The polypeptide chain therefore folds so that its hydrophobic side chains are buried and its polar, charged chains are on the surface.  Tertiary Structure Stabilizing Bonds: Four types of bonding interactions between "side chains” are involved in protein folding: Covalent bond – Disulfide bonds Non covalent bond – Hydrophobic interactions – Electrostatic interaction – Hydrogen bonding – Salt bridges/ionic bond  Tertiary Structure Stabilizing Bonds ü Ionic bonding in the interior is rare because most charged amino acids lie on the protein surface. § Covalent bonds are the strongest chemical bonds contributing to protein structure. Covalent bonds arise when two atoms share electrons. § Disulfide bonds - Formed by oxidation of the thiol groups on cysteine. Important for protein folding and stability, usually extracellular protein. § Ionic bonds are formed as amino acids bearing opposite electrical charges are juxtaposed in the hydrophobic core of proteins. § Ionic bonding in the interior is rare because most charged amino acids lie on the protein surface. § Groups on the surface of a protein that are capable of forming hydrogen bonds or ion dipole bond increase the aqueous solubility of the protein.  Tertiary Structure Stabilizing Bonds: Hydrophobic Interactions for Protein Folding: ü Non-polar molecules to aggregate in aqueous solutions in order to separate from water § The hydrophobic effect is the desire for non-polar molecules to aggregate in aqueous solutions in order to separate from water. § Hydrophobic bonding forms an interior, hydrophobic protein core, where most hydrophobic side chains can closely associate and are shielded from interactions with solvent H2O.  Quaternary Structure: Quaternary structure of deoxyhemoglobin § Quaternary structure is the combination of two or more polypeptides chains, to form a complete unit. Each polypeptide chain in such a protein is called a subunit. § The interactions between the chains are not different from those in tertiary structure, but are distinguished only by being interchain rather than intrachain. § Simplest form: dimer with two identical subunits Common form: consists of more than two different subunits § Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. § Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-", for example, a heterotetramer, such as the two alpha and two beta chains of hemoglobin.  Protein Folding: § Protein folding is the process by which a protein structure assumes its functional shape or conformation. § Each protein exists as an unfolded polypeptide when translated from a sequence of mRNA to a linear chain of amino acids. § Amino acids interact with each other to produce a well-defined 3D structure, the folded protein, known as the native state. § While the process is trial and error, the result is usually a protein that can exist in a low-energy state.  Protein folding is a spontaneous process “hydrophobic collapse” hydrophilic amino acids hydrophobic amino acids § An uncoiled polypeptide spontaneously attains the proper three dimensional shape so that it can function properly. § Protein folding depends on the amino acid sequence and the physicochemical properties of the amino acid residues. § Folding occurs due to a process known as hydrophobic collapse, in which the hydrophobic residues spontaneously collapse into the interior of the protein molecule away from the aqueous exterior. Non-polar side chains in the inside Polar side chains on the outside Chemical interactions between amino acids § This is an energetically favored process (laws of thermodynamics and chaperones’ help). § The “minimal frustration principle”  The protein searches for its random coil lowest energy conformation Lowest free energy Most stable Most functional Energy © Adam Steinberg “native state” § The folding process can be viewed as a kind of free-energy funnel. § Because the side chains of each amino acid along the peptide backbone have different physical chemical properties (e.g., length, bulk, polarity, charge, hydrophobicity, etc.) they will arrange themselves such that the final shape is one that has the lowest possible energy state within an aqueous environment. § From a thermodynamic perspective, the polypeptide “random coil” quickly searches for the conformation (shape) that achieves the lowest energy state. § The shape with the lowest free energy is the most stable and most functional. This is called the “native state.” § Formation of secondary structures like alpha helices and beta-sheets through hydrogen bonding help to minimize the energy. Other hydrophobic interactions and electrostatic interactions aid in bringing the random coil into a highly ordered, 3-dimensional, low energy tertiary or “native state.” § On occasion, a conformation will get stuck in a local energy minimum that is not ideal and may not function properly. Such “misfolded” conformations are less functional.  Pharmacological Chaperones Molecular Pharmacological Chemical § Misfolded proteins are usually retained by the RER and then degraded § Pharmacological chaperones are small molecules that are supposed to act in the RER and assist in correct protein folding  When folding goes wrong § Protein misfolding is now implicated in the progression of hundreds of diseases. § Most misfolding stems from genetic mutations like single nucleotide polymorphisms (SNPs). § Protein misfolding is involved in the majority of diseases not caused by a conventional infectious agent. § Protein folding can cause disease as a result of: Improper protein degradation - CFTR Improper protein localization - α1-antitrypsin Dominant-negative mutations – keratin in e.bullosa. Gain of toxic function – APOE4 Amyloid accumulation – amyloidosis § Improper degradation: Although cellular degradation systems, such as autophagy, are essential for preventing the accumulation of non-functional misfolded proteins, they sometimes cause disease by being overactive, degrading proteins that, although mutant, retain some functionality, e.g. cystic fibrosis, which is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane chloride channel. § Improper localization: Because many proteins that localize to specific organelles must fold correctly in order to be trafficked properly, mutations that destabilize the correct fold can lead to improper subcellular localization. This can result in dysfunction via both loss of function of the protein at its appropriate location as well as gain-of-function toxicity if it accumulates in an incorrect location, e.g.α1-antitrypsin, a secreted protease inhibitor that, when mutated, leads to emphysema. § Dominant-negative mutations: Occurs when a mutant protein negatively effecting the function of the wild-type protein, causing a loss of protein activity even in a heterozygote. In epidermolysis bullosa simplex, an inherited connective tissue disorder, mutant forms of the keratin proteins and lead to severe blistering of the skin in response to injury. Keratin forms long intermediate filaments with both wildtype and mutant versions of the protein, which does not function properly. § Gain of toxic function: Protein conformational changes can also cause dominant phenotypes by causing a protein to acquire a conformation that contributes to toxicity. One example is apolipoprotein E (APOE), a lipid transport molecule. The polymorphism in APOE4 stabilizes an altered conformational fold of the protein and changes its lipid affinity, thus disrupts mitochondrial function and impairs neurite outgrowth. § Amyloid accumulation. The ability of stable amyloid fibers – insoluble fibrous protein aggregates – to accumulate and contribute to a variety of diseases. These range from neurodegenerative disorders (including Parkinson’s disease and Huntington’s disease) to amyloidosis (such as familial amyloid polyneuropathy).  Genetic mutations lead to misfolding Salt bridge + Wild type (left) and mutated (right) form of lamin A. § Protein function depends on the chemical properties of amino acid side chains and their location relative to each other § Therefore, protein function depends on its correct folding  Amyloids aggregate into plaques and fibrils q sporadic, acquired or inherited © Elsevier Certain proteins can form amyloid structures, which are layers of β-sheets not found in the native conformation. These sheets aggregate to form plaques and/or fibrils that can lead to altered cellular function and cell death. Examples: Neurodegenerative disorders (ALS, Parkinson’s disease), Prion diseases (Creutzfeldt-Jakob disease), Cataracts, others. § Certain proteins can form amyloid structures = layers of β-sheets Spontaneous/sporadic: no known cause Acquired: for instance in older cells misfolded proteins may not be detected and degraded that soon Inherited: mutations in a protein can lead to amyloid formation at an early age § The formation of amyloids is not well understood and is more complex than only protein misfolding. Other proteins, cellular environment, protein concentration all play a role § Amyloids form plaques that can lead to reduced cellular function and cell death § Examples: Diabetes (destroying insulin-producing cells) and neurodegenerative disorders (Alzheimer’s disease and Prion diseases, many proteins are expressed throughout the body but only form amyloids in the CNS) § Amyloids do not always cause disease.  ? amyloid precursor protein (APP) Protein Misfolding: Alzheimer’s Disease § β-amyloid proteins are formed from the amyloid precursor protein (APP), a transmembrane protein expressed on neurons, by proteolytic cleavage § The role of these plaques in the Alzheimer disease process is not known, there are more variables § Most of the data is now controversial as the authors accepted that they manipulated the data.  Protein Misfolding: Mad Cow Disease PrPSC GI Transmissible Spongiform Lymphoid colony Encephalopathies Sympathetic nerves Infect PrPc PrPC PrPSC Prion: proteinaceous infectious particle Creutzfeldt-Jakob disease § In prion disease, the infective agent is an altered version of a normal prion protein that acts as a “template” for converting normal protein to the pathogenic conformation. § Prion stands for proteinaceous infectious particle § Normal prions (PrPC ) contain mostly α-helices and a few β-sheets and are anchored to the cellular membrane of many cell types § Their function is not known § PrPSc are misfolded prion proteins (“Sc” comes from scrapie, a prion disease in sheep) that contain many more β-sheets than PrPC § PrPSc form amyloid sheets that are very resistant to proteolytic cleavage and denaturation by heat § Even though prions are misfolded proteins, when consumed, they can easily survive the harsh conditions of the GI tract. § Prions traverse the intestinal epithelium where they colonize lymphoid tissues of the gut. They can replicate in follicular dendritic cells that express PrPC. § Prions (PrPSC) can travel up afferent sympathetic nerves from the gut to the brain where they can “infect” normal PrPC proteins.  Protein Misfolding: Diabetic Cataract Crystallins are a major protein component of the lens of the eye. In their native conformation, they form a transparent structure that allows light to pass through the lens. Crystallins are also prone to forming amyloid fibers, which cause cataracts that scatter light, clouding vision.  Protein Denaturation: Denaturation is a process in which a protein loses its native shape due to the disruption of weak chemical bonds and interactions, thereby becoming biologically inactive. When protein is denatured it losses its function. Examples: A denatured enzyme ceases/stops its function. A denatured antibody no longer binds to its antigen. A denatured milk proteins losses its biological activity § A protein’s function depends on its 3D structure. § Loss of structural integrity with accompanying loss of activity is called denaturation. § Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape. § Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non- polar hydrophobic interactions. which may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein. § Proteins can be denatured by: heat or cold pH extremes organic solvents chaotropic agents: urea and guanidinium hydrochloride Mechanism of protein denaturation: Various agents which causes denaturation of proteins: Physical agents: Heat Violent shaking or agitation Hydrostatic pressure UV radiation Chemical agents: Acids and alkalis Organic solvents Salts of heavy metals Chaotropic agents Detergents Altered pH Denaturation by heat: Most proteins can be denatured by heat, which affects the weak interactions in a protein (primarily hydrogen bonds) in a complex manner. If the temperature is increased slowly, a protein’s conformation generally remains intact until an abrupt loss of structure and function occurs over a narrow temperature range. During cooking, this stress causes denaturation which is typically as heat and ultimately proteins gets coagulated. Denaturation by acids and bases: Acids and bases disrupt salt bridges held together by ionic charges. Double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system when acidic gastric juices coagulates proteins.  Denaturation by organic solvents: (e.g. alcohol, acetone, diethyl ether) Alcohol disrupts the hydrogen bond between amide groups in secondary structure as well as between side chains in tertiary structure in proteins. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains. § A 95% alcohol solution coagulates the protein on the outside of the cell wall of bacteria and prevents any alcohol from entering the cell. § Therefore, 70% alcohol solution is used as a disinfectant. § This concentration of alcohol is able to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell. § This is common practice in GMP facilities and research labs.  Denaturation by detergents: Detergents are amphipathic in nature having both hydrophobic side and a hydrophilic side. Proteins have hydrophobic and hydrophilic sides, the detergent is attracted to these and forces the protein apart. A protein's 3-D structure is partially created by hydrophobic and hydrophilic interactions to itself, the detergent substitutes this self bonding with detergent-amino acid bonding. Furthermore, detergent is a salt and breaks up positive and negative interactions of the 3-D shape as well and denatures the proteins. Denaturation by altered pH: The ionization rate of the functional groups in protein amino acid chains depends on the functional group and the pH. A high concentration of hydrogen ions (low pH) will result in more groups being protonated. Charged groups will tend to move towards the surface of the proteins and uncharged groups tend to move inwards.  Denaturation by salts of heavy metals : The heavy metal salts usually contain Hg2+, Pb2+, Ag1+ Ti1+, Cd2+ and other metals with high atomic weights. Since salts are ionic in nature they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt complex. Heavy metals may also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will also lead to the denaturation of proteins. These reactions are used for their disinfectant properties in external applications, such as: silver nitrate is used in the treatment of nose and throat infections or cauterize wounds as well as to treat diseases such as: auranofin (gold salt) for rheumatoid arthritis