Biochem (Module 3)

Classification of amino acid side chains

-20 amino acids are groups into different classifications according to polarity and structural features of the side chains -Each amino acid has both a three letter and one letter designation

Protein families and superfamilies

-A family of proteins is composed of homologous proteins related to the same ancestral protein -Paralogs are groups of proteins within the same species with similar, but not identical, structure and function that have evolved from the same gene after the gene was duplicated -Paralogs of a protein family are considered to be different proteins and have different names because they have different functions; they are all present in the same individual -Large families of homologous proteins that share one defining region (or 'domain') are called a superfamily, which is then divided into families of proteins that share other similar structures

Amino acid modifications

-After synthesis of proteins, a few amino acids are posttranslationally modified -Modifications change the structure of one or more amino acids on a protein in a way that may serve a regulatory function, target or anchor the protein in membranes, enhance a protein's association with other proteins or target it for degradation -Addition of a carbohydrate (glycosylation) -Addition of a lipid -Regulatory modification (phosphorylation, acetylation, ADP-ribosylation) -Carboxylation or oxidation of amino acids -Selenocysteine -Found in a few enzymes and is required for their activity -Not a posttranslational modification, but a modification of serine that occurs while serine is bound to a unique tRNA ·-A selenium atom replaces the hydroxyl group of serine; it is then inserted into the protein as it is being synthesized ·-Found in 25 human proteins, many involved in cellular metabolism and regulation of overall metabolic rate

Aliphatic, polar, uncharged amino acids

-Amino acids with side chains that contain an amide group (aspargine and glutamine) or a hydroxyl group (serine, threonine) -Asparagine and glutamine are amides of the amino acids aspartate and glutamate -These side chains can form hydrogen bonds with water or other side chains of amino acids -Very hydrophilic, often found at surface of water-soluble globular proteins

Charged amino acids

-Aspartate and glutamate have carboxylic acid groups that carry a negative charge at physiologic pH (acidic) -Histidine, lysine, and arginine have side chains containing nitrogen that can be protonated and positively charged at physiologic or lower pH values (basic) -Amino acid side chains change from uncharged to negatively charged (acidic), or positively charged to uncharged as they release protons (basic) -In proteins, only the side chains, and the terminal amino and carboxyl group have dissociable proteins §Positive charges on basic amino acids enable them to form ionic bonds with negatively charged groups on other side chains or to the phosphate groups of coenzymes

Secondary structure: α-helix

-Common secondary structural element found in globular proteins, membrane-spanning domains, and DNA-binding proteins -Contain repeating elements formed by hydrogen bonding between atoms of the peptide bonds -Each peptide bond is connected by hydrogen bonds between each carbonyl oxygen atom and the amide hydrogen of an amino acid residue 4 residues further down the chain or between the amide hydrogen and the carbonyl oxygen 4 residue further ahead -Core is tightly packed; side chains project backward and outward from the helix, avoiding steric hinderance with the backbone and each other -Proline cannot form necessary bond angles to fit within α-helix and is a 'helix breaker'

Tertiary structure: transmembrane proteins

-Contain membrane-spanning domains and intra- and extracellular domains on either side of the membrane -Include ion channel proteins, transport proteins, neurotransmitter receptors, and hormone receptors -They may contain ligand binding sites on the extracellular side and signal transdusing domains on the intracellular side -May contain modified amino acids (glycosylated show here) for signal interactions extracellular or phosphorylation domains intracellular for signal transduction

Sulfur containing amino acids

-Cysteine has a sulfhydryl group -Sulfhydryl group has a pKa of 8.4, so primarily protonated (and uncharged) at physiologic pH -In proteins, formation of cystine disulfide bonds between appropriate cysteine amino acids plays an important role in holding two polypeptide chains (interchain disulfide bonds) or two different regions (intrachain disulfide bonds) together -Methionine is nonpolar with a large, bulky sulfur-containing side chain that is hydrophobic -Cannot form sulfide forms, but plays role in metabolism by transferring methyl group to other compounds -Because of this ability, it is often the first amino acid in a protein

General structure of amino acids

-Every amino acid contains a carboxylic acid group, an amino group attached to the α-carbon, a hydrogen atom, and a chemical group that is called a side chain -At physiologic pH (7.4), the free amino acid exists as a zwitterion -All can form hydrogen bonds and are soluble in water -Glycine is the exception because it has two hydrogen groups; all other amino acids have 4 different groups bound to alpha carbon -Zwitterion means it has both a positive and negative charge; pKa of carboxyl group is ~2 (so 99% deprotonated at 7.4) and pKa of amino group is 9.4 (so almost all protonated)

Nonpolar, aliphatic

-Glycine is hard to classify, because it has two protons (one as R group) -Alanine and the branched amino acids (valine, leucine and isoleucine) have bulky, nonpolar, aliphatic side chains that exhibit a high degree of hydrophobicity -Do not hydrogen bond and will form hydrophobic cores within proteins -Proline contains a ring that kinks the peptide backbone (often initiates turns in proteins) -Glycine is the most compact amino acid (causes the least amount of steric hinderance) and is often found in bends or other parts of the protein where bulky amino acids will affect structure too much -Aliphatic means (open chain hydrocarbons

Aromatic amino acids

-Grouped together because they all contain ring structures with similar properties, but polarities differ a great deal -Aromatic ring is a six member carbon-hydrogen ring with 3 conjugated double bonds -Hydrogens do not form H-bonds; substituted functional groups determine polarity and activity

Secondary structure: β-sheets

-Hydrogen bonding occurs between regions of separate neighboring polypeptide strands aligned parallel to each other -Notice contrast to α-helix, where the hydrogen bonds occur within the sae same strand -Optimal hydrogen-bonding occurs when the sheet is bent (pleated) -Antiparallel strands are often part of the same protein looping back on itself and usually have hydrophobic side and hydrophilic side -Parallel sheets tend to have hydrophobic residues on both sides of the strand

Building proteins

-In proteins, amino acids are joined by peptide bonds between the carboxylic acid of one amino acid and the amino group of an adjacent amino acid -Side chains 'stick out' from peptide backbone and interact with other polar molecules; determine folding -Chemical properties of the side chains determine how the protein folds, how it binds specific ligands, and how it interacts with its environment -The second picture is the active site of an enzyme involving 2 tyrosines (Y) and one cysteine (C) -Each chain will have an amino terminal and a carboxyl terminal -Protein sequences are ALWAYS written from N-terminal to C-terminal

Requirements for protein structure

-Must create binding sites that is specific for one molecule or group of molecules -The binding sites define the role of the proteins -Must exhibit a degree of flexibility and rigidity appropriate for its function -Rigidity is essential to create specific binding sites and stability, but flexibility allows the protein to fold appropriately and adapt to binding other proteins and small molecules -Must have an external structure appropriate to environment the protein inhabits -E.g., surface needs polar amino acids for aqueous environments, nonpolar amino acids for hydrophobic environments -Conformation must be stable with little risk of refolding into nonfucntional shape -Structure must be able to be degraded when protein is damaged or no longer needed

Myoglobin and hemoglobin

-Myoglobin and the different chains of hemoglobin are paralogs and members of the globin family -Myoglobin, an intracellular heme protein present in most cells stores and transports O2 to mitochondria, is a single polypeptide chain with one heme oxygen-binding site -Hemoglobin is composed of 4 globin chains, each with its own heme oxygen-binding site, present in RBCs—transports O2 from lungs to tissues -Gene for myoglobin is assumed to have evolved from gene duplication of the alpha chain of hemoglobin, which itself evolved from a duplication of the beta chain of hemoglobin

Charged amino acids part 2

-Positive charges on basic amino acids enable them to form ionic bonds with negatively charged groups on other side chains or to the phosphate groups of coenzymes -Lysine and arginine side chains often form ionic bonds with negatively charged compounds bound to protein binding sites (phosphate groups of ATP) -Acidic and basic amino acid side chains also participate in hydrogen bonding and the formation of salt bridges -The acidic amino acids lose a proton from their side chains at a pH of approximately 4 and are negatively charged at pH 7.4 -Basic amino acids change from positively charged to neutral at their pKa ·-Lysine and arginine have pKa values >10, so they are fully protonated (positive charge) at physiologic pH ·-Histidine has a pKa value of 6, so only a portion of side chains carry a positive charge at physiologic pH

Protein structure

-Primary is the linear sequence of amino acids joined through peptide bonds -Secondary is the recurring structure that form in short localized regions of the polypeptide chain (alpha-helices, etc.) -Tertiary is the overall 3-D structure that includes the totality of the secondary structure - The secondary and tertiary structure possible is limited by the fact that the carboxyl and amide groups must remain planar with the alpha carbon and R group having some rotation capability—steric constraints maximize the distance between atoms in different amino acid side chains -Quartenary is the association of polypeptide subunits in a geometrically specific manner -The forces driving these interactions are primarily non-covalent (van der Waals, hydrogen-bonding, ionic interaction, etc)

Intro to proteins

-Proteins have many functions in the body -The unique characteristics of proteins are dictated by the primary structure ( the linear sequence of amino acids) -Primary structure determines how the protein folds and how it interacts with other molecules in the cell to perform its function -Primary structures of all human proteins are synthesized from 20 amino acids arranged in a linear sequence determined by the genetic code

Tissue specific isoforms

-Proteins that differ in primary structure and properties from tissue to tissue, but retain essentially the same function, are called tissue-specific isoforms -Creatine kinase is composed of two subunits; depending on the tissue, different homologous subunits comprise CK -M form is produced in skeletal muscle (MM); B form is produced in the brain (BB); Heart produces both (MB and each of the homodimers; Two more isozymes found in mitochondria -Not clear what the advantage of the different forms is -Proteins that are involved in the response to hormones often exist as several tissue-specific isoforms that help different tissues respond differently to the same hormone -Adenylyl cyclase (9 different adenylyl cyclase isoforms coded by different genes in different tissues) -Different isoforms help cells respond differently to the same hormone

Variations in protein structure

-The amino acid sequence of a normal functional protein can vary somewhat among individuals, tissues, or during development -These variations are tolerated if they occur in non-critical regions (variant regions) of the protein or if they confer a selective advantage (Red boxes) -In contrast, regions that form binding sites or are critical for 3-D structure are usually invariant regions (all gray shaded amino acids) -Conservative changes mean one amino acid has been substituted for an amino acid in the same group (polar for polar, charged for same charged, etc); non-conservative means that the nature of the amino acid has changed

Protein structure-function relationship

-The overall conformation of the protein, including the positioning of the amino acid side chains in three-dimensional space, determines the function of the protein -Structural classifications -Globular proteins (resemble irregular balls; soluble in aqueous solutions); includes most DNA-binding proteins -Fibrous proteins (linear, arranged in a single axis, repeating structure) -Transmembrane proteins (one or more regions aligned to cross the lipid membrane; have hydrophilic portions exterior to membrane and hydrophobic portions interior to membrane)

Secondary structure

-Two most common secondary structures are the α-helix and the β-sheet -Contain repeating elements formed by hydrogen bonding between atoms of the peptide bonds -Other regions of the polypeptide chain form nonregular, nonrepetitive secondary structures such as loops and coils

Polymorphism in protein structure

-Variations of an allele that occur with frequency in a population are referred to as polymorphisms -Variations among humans usually arise as a result of mutations in the DNA that are passed generation to generation; they may be beneficial, detrimental, or conservative (no consequence) -In humans, almost one third of the genetic loci seem to be polymorphic -Sickle cell allele is an example of a stable polymorphism; its persistence in endemic areas is probably the result of selection for the heterozygous mutant phenotype, which confers protection against malaria -When a particular variation is found with a frequency >1%, it is considered stable

Agents that affect O2-binding

•2,3-bisphosphoglycerate •Glycolytic intermediate formed in RBCs; binds in central cavity formed by four subunits, increasing energy required to facilitate O2 binding; O2 is more readily released into tissues when hemoglobin is bound to 2,3-BPG; RBCs can modulate hemoglobin affinity for O2 by altering rate of synthesis or degradation of 2,3-BPG •Proton binding •Binding of protons lowers hemoglobin affinity for oxygen; pH of blood decreases as it enters the tissues (and proton concentration increases), because metabolic CO2 is converted to carbonic acid by carbonic anhydrase in RBCs; dissociation of carbonic acid produces protons that interact with amino acids on hemoglobin and trigger conformational changes that promote the release of oxygen •In the lungs, oxygen binds to hemoglobin (high PO2), causing a release of protons, which combine with bicarbonate to form carbonic acid; decrease of protons results in an increase in pH, which drives carbonic anhydrase to work in reverse, cleaving carbonic acid into water and CO2 and the CO2 gets expelled •CO2 (a little bit)

nonrepetitive secondary structures

•Bends, loops, and turns do not have repeating elements of hydrogen bond formation •Ribbon diagram of a globular domain •Collectively, the totality of the of the secondary structures makes up the tertiary structure

Myoglobin and hemoglobin: structure-function relationships part 5

•Changes in tertiary structure that take place when O2 binds triggers cooperativity in oxygen binding to hemoglobin •Change in conformation is described as T for tense (low affinity for oxygen) and R for relaxed (high affinity for oxygen) •Binding rate of O2 to the first subunit is very low (must overcome energetics, occurs only at higher PO2) when hemoglobin is in the T state; after binding of the first oxygen, subunit enters the R state and the conformational change will trigger all other subunits to be in the R state, as well—this greatly increases binding rate of other subunits •This is why curve is sigmoidal—rate to get the first oxygen on is low, but once PO2 is high enough to overcome the energetics, all other subunits become more receptive and oxygen binding increases exponentially •At the other end (low PO2), oxygen will unbind, causing a return to the T state in one subunit, which increases the favorability of the other subunits unbinding their oxygen, too

Protein denaturation

•Denaturation through nonenzymatic modification of proteins •Glucose concentration drives glycosylation and oxidation of amino acids, resulting in loss of function and inability of the cells to degrade the proteins •These proteins aggregate and contain nonfunctional proteins (these accumulate with age) •Protein denaturation by temperature, pH, and solvent •Disrupt ionic, hydrogen, and hydrophobic bonds and interactions •pH disrupts interactions occurring with carboxylate groups (low pH) or amino groups (high pH) •Temperature increases vibrational and rotational energy energies in bonds, affecting their stability and ability to maintain 3-D shape •Amphipathic solvents can dissolve proteins by creating or disrupting hydrophobic interactions

Protein folding

•Every molecule of the same protein folds into the same stable 3-D shape (native conformation) •Primary sequence dictates folding patterns into 3-D conformation •Some denatured proteins, even those with cofactors, can refold if the environmental conditions are correct •Not all proteins can achieve their native conformation without help, however •As a protein folds to achieve its low-energy state (the most energetically favorable fold), it may have to pass through high energy conformations that slow the process (kinetic barriers) •These kinetic barriers can be overcome by heat-shock proteins (also called chaperonins) which use ATP to assist in the folding process

Tertiary structure: folds

•Folds are patterns of 3-D structure that are found in many proteins •A characteristic activity is associated with each fold, such as ATP binding and hydrolysis (actin fold) or NAD+ binding (nucleotide binding fold) -Actin-folds are named after the protein in which the domain was first identified and is found in diverse proteins like heat shock proteins and hexokinase; there is little sequence similarity between these proteins, but the domains are superimposable and have enough identity to suggest a common evolutionary ancestor

Myoglobin and hemoglobin: structure-function relationships

•Myoglobin is found within heart and skeletal muscle and captures oxygen to deliver to mitochondria for fuel oxidation in energy •Hemoglobin is found in red blood cells and carries oxygen from lungs to your tissues and CO2 from your tissues to your lungs •Both oxygen-binding proteins with very similar primary structure •Myoglobin is a globular protein composed of a single polypeptide chain with one oxygen-binding site •Hemoglobin is a tetramer composed of 2 different types of subunits (2 α- and 2 β-polypeptide chains; 2 αβ-protomers) •Each subunit has a strong sequence homology to myoglobin and each contains an oxygen binding site •Tetrameric structure of hemoglobin facilitates saturation with O2 in the lungs and release of O2 in capillary beds

Prions part 2

•PrPC is primarily α-helical, whereas PrPSc is primarily β-sheet •This difference favors the aggregation (stacking) of PrPSc into multimers that can't be degraded •High kinetic barrier usually prevents refolding of PrPC into PrPSc, but if PrPSc is ingested, this stabilized the refolding of PrPC and triggers the formation of aggregates

Prions

•Prions are (potentially) infectious proteins that cause neurodegenerative disease by acting as a template to misfold other cellular prions proteins to a form that cannot be degraded •They may be acquired through infection or from sporadic or inherited mutations •Prion proteins (PrPc) are normally found in the brain and is encoded by a gene that is a normal component of the human genome •The disease causing prion (PrPSc) has the same primary sequence, but is (mis)folded into a form that generates multimer aggregates that are resistant to digestion

quartenary structure

•Refers to the association of individual polypeptide subunits in a geometrically and stoichiometrically specific manner •Dimers, trimers, tetramers, oligomers come together to make one functional protein •Homo- or hetero- refers to whether the subunits are identical or different , respectively •Protomer is a unit structure composed of nonidentical subunits (imagine an protein formed of A2B2, where one AB pair is a protomer) •Oligomer is a multi-subunit protein comprised of identical subunits (A6) •Multimer is a generic term for a complex with different types of subunits (A3B3) •The contact points of the subunits involve interactions between nonpolar side chains, hydrogen bonds (intermolecular), ionic bonds or salt bridges •They are rarely covalently connected, but occasionally, they will have disulfide bonds between subunits -Multiple subunits increases stability and increases the number of possible interactions (more difficult to fold and unfold) -May enable proteins to act cooperatively between subunits binding ligands (hemoglobin) or to form binding sites with high affinity for large moleucles -May have different activities and cooperate in a common function

Myoglobin and hemoglobin: structure-function relationships part 3

•Tertiary structure of myoglobin contains 8 α-helices connected by short coils (globin fold) •This creates a hydrophobic O2-binding pocket that contains a tightly bound heme with an iron atom in its center •Heme interacts with protein ionic interactions with polar side chains of myoglobin amino acids and hydrophobic interactions with hydrophobic side chains of myoglobin amino acids •There are about 16 interactions between heme and myoglobin amino acids, positioning the prosthetic group perfectly within the protein -A protein with its prosthetic group is called a holoprotein; without the prosthetic group is called an apoprotein -Prosthetic group does not dissociate until the protein is degraded

Tertiary structure

•The pattern of the secondary structural elements folding into a three-dimensional conformation •Flexible and dynamic allowing for ions and water to diffuse through the structure without unfolding the protein •Provides alternate conformations for ligand binding •Tertiary structure is often composed of independent regions called structural domains •Often can identify domains visually from the 3-D structure (next slide) •Each domain is formed from a continuous sequence of amino acids in the polypeptide chain that are folded into a 3-D structure independently of the rest of the protein and two domains are connected through simple structures like loops •The structural features (and sometimes functions) can be discussed independently of another domain in the same protein

Myoglobin and hemoglobin: structure-function relationships part 2

•When PO2 is high, both myoglobin and hemoglobin are saturated with oxygen •At lower PO2 (such as in tissues), myoglobin binds O2 better than hemoglobin •Thus, as hemoglobin is releasing oxygen into tissues, myoglobin can still pick it up and deliver to the cytochrome oxidase in the electron transport chain found in mitochondria •Myogoblin curve is hyperbolic (oxygen added consistently relative to PO2), whereas hemoglobin curve is sigmoidal (oxygen added inconsistently until some particular PO2 is achieved, and then oxygen saturation increases) -Think about function, myoglobin just needs to bind oxygen until a compound with much higher affinity for oxygen comes to rip it away (cytochrome oxidase); hemoglobin must bind oxygen where it is high and then deposit it in low oxygen tissues and not rebind it if the oxygen is not immediately snatched up—this means you want to actively discourage oxygen binding at low PO2

Myoglobin and hemoglobin: structure-function relationships part 4

•Within both myoglobin and hemoglobin, O2 binds directly to Fe2+ atom on one side of the ring •The Fe2+ atom is able to coordinate with 6 different ligands: 4 are within the porphyrin ring of heme, one (top) is the nitrogen on the (proximal) histidine of myogoblin and hemoglobin, the other is to O2, CO, or empty -Proximal histidine is sterically hindered by the rest of the porphoryin ring, so when it binds to iron, it pulls the iron above the plane of the ring; when oxygen binds, it pulls the iron back down, and changes the position of the helix