Proteins: Structural Complexity and Pathology

• Proteins have multiple levels of structural complexity

Levels of Structural Complexity

• The primary structure is the linear amino acid sequence held by peptide bonds
• Primary structure determines higher-order structures and any disulfide bonds
• The genetic code correlates with the primary structure and contains specifications for all protein structure levels
• Amino acid sequences are read from left to right, starting with the amino terminal
• An example of a tetrapeptide is alanylaspartylglycylleucine
• Polymerization of amino acids yields a linear molecule called a polypeptide
• Nomenclature indicates the number of amino acids, such as dipeptide (two amino acids) or oligopeptide (few amino acids)
• The properties of a polypeptide are determined by amino acid side chains

Pathology

• Mutations in hemoglobin can cause disease
• Sickle cell hemoglobin (HbS) and hemoglobin C (HbC) both have single amino acid substitutions at residue 6 of β-globin
• HbS has valine (nonpolar) instead of glutamate (polar), while HbC has lysine (polar) instead of glutamate (polar)
• These primary structure changes affect quaternary structure, leading to serious sickling attacks in HbS
• HbC causes mild chronic hemolytic anemia

Secondary Structure

• Secondary structure involves regular, extended structures stabilized by hydrogen bonding between peptide bonds
• Side chains can influence the type and stability of secondary structures
• Two main types of secondary structures are α-helix and β-pleated sheet
• α-helix is a right-handed helix with peptide bonds inside and side chains extending outward
• It is stabilized by hydrogen bonds parallel to the helix axis between amino and carbonyl groups every fourth peptide bond
• Proline, lacking a free hydrogen, is a "helix breaker"
• α-helices are common in globular proteins and some fibrous proteins like α-keratin
• β-pleated sheet structure consists of extended, adjacent polypeptide sequences stabilized by hydrogen bonds
• Adjacent chains can be parallel or antiparallel
• β-structures are found in 80% of globular proteins and silk fibroin

Microbiology

• Prion diseases are caused by prions (PrPSc)
• PrPSc are formed from normal neurologic proteins (PrP)
• Prion diseases include Creutzfeldt-Jakob disease, kuru, scrapie, and bovine spongiform encephalopathy
• Contact between PrP and PrPSc converts PrP's secondary structure from α-helical to β-pleated sheet
• The altered protein forms filamentous aggregates that damage neuronal tissue
• PrPSc is highly resistant to heat, ultraviolet irradiation, and proteases

Supersecondary Structure and Domains

• Supersecondary structures (motifs) are characteristic combinations of secondary structures, 10-40 residues long, recurring in different proteins
• Motifs bridge the gap between secondary and tertiary structure, with the same motif performing similar functions in different proteins
• The four-helix bundle motif provides a cavity for enzymes to bind prosthetic groups or cofactors
• The β-barrel motif can bind hydrophobic molecules like retinol inside the barrel
• Motifs can be mixtures of α and β conformations
• Motifs can have specific ligand binding functions or contribute to domain structure
• Primary structure is subdivided into domains, typically 25-300 residues long
• Within a polypeptide, domains independently fold into stable configurations
• Exons are regions within structural genes that code for domains
• A domain can consist of one or more secondary structure motifs
• Domains contribute to protein's 3-D structure, but do not describe complete (tertiary) structure
• Polypeptide bending interrupts regular structures in domains
• α-Helices bend at proline residues
• β-Structures bend at β-turns, looping back into domain structure

Tertiary Structure

• Tertiary structure is the complete 3-D structure of a polypeptide, stabilized by side chain interactions and disulfide bonds in extracellular proteins
• Folding brings distant sequences together into a stable structure
• Hydrophobic side chains are found in the interior of soluble proteins to minimize exposure to water
• Hydrophilic amino acids that can form hydrogen bonds to water are at the surface
• Integral membrane proteins have hydrophobic groups spanning the membrane and hydrophilic groups on the surface
• Native conformation is the most stable structure under physiologic conditions
• Four side chain interactions that stabilize the native conformation include:
  • Hydrophobic interactions: Hydrophobic side chains are forced together at the interior of proteins
  • Van der Waals forces: Non-specific attractions develop based on proximity of interacting atoms
  • Electrostatic bonds: Oppositely charged side chains form salt bridges
  • Hydrogen bonds: Polar groups share a partial positive charge
• Some proteins require a prosthetic group for functionality, where the apoprotein lacks the prosthetic group
• Holoprotein includes the prosthetic group, attached covalently (e.g., biotin to lysine) or noncovalently (e.g., heme)
• Tertiary structure of fibrous and globular proteins is adapted to their biological role
• α-keratin is a multiunit elastic fibrous protein with protofibrils as its basic unit
• Protofibrils consist of four right-handed α-helices wound in a left-handed supercoil
• Microfibrils are coiled from protofibrils and cross-linked by disulfide bonds
• Silk fibroin, an inelastic fibrous protein, is composed of antiparallel β-pleated sheets, and is highly resistant to protease digestion
• Disulfide bonds stabilize native conformation in the extracellular space, formed by protein disulfide isomerase during polypeptide folding

Quaternary Structure

• Quaternary structure is the subunit composition of a protein
• Polypeptide subunits associate specifically to form a functional oligomer (dimer, tetramer, etc.)
  • Heteromeric: composed of different subunits, each from a different gene
  • Homomeric: composed of the same monomer unit, all are produced by the same gene
• Quaternary structure is held together by noncovalent bonds between complementary surface regions
• Acidic and basic side chains can form salt linkages
• Subunits can dissociate due to the same weak forces that stabilize tertiary structure
• Covalent stabilization can occur via interchain disulfide bonds
• Subunit contact allows interaction where a shape change in one subunit induces a change in another subunit
• Multiple subunits are affected by single ligand binding

Neuroscience

• β-Amyloid protein is associated with neurodegenerative diseases, such as Alzheimer disease (AD)
• AD involves loss of function and death of neurons, leading to loss of cognitive function and memory
• Pathologic changes with AD form neuritic plaques and neurofibrillary tangles
• Neuritic plaques contain β-amyloid protein from proteolytic conversion of the neuronal β-amyloid precursor protein (APP)
• β-Amyloid deposits in neurons are neurotoxic

Pathology

• Heinz bodies are composed of denatured hemoglobin that has become oxidized in RBCs
• Denatured hemoglobin forms visible aggregates on the RBC membrane
• Heinz bodies form with high oxidative stress or unstable hemoglobin variants

Key Points About Levels of Structural Complexity

• Protein function is dependent on its stable native conformation
• Primary structure is the linear amino acid sequence with planar peptide bonds
• Secondary structure is a regular extended structure limited to α-helix and β-structure
• Supersecondary structure motifs are characteristic associations of secondary structure
• Domains are independent 3-D structures of supersecondary structure motifs that perform specific functions within a protein

Denaturation

• Denaturation is the disruption and loss of native conformation, leading to a loss of biological activity
• Denaturation is caused by non-physiologic conditions:
  • Extremes in pH or ionic strength
  • Detergents
  • Increased temperature
  • Reaction with heavy metals
• As conditions change, higher-order structure gradually disrupts
• Primary structure remains unaffected
• Denatured polypeptide becomes randomized and aggregates, forming an insoluble precipitate
• Denatured proteins are more susceptible to digestion by proteolytic enzymes
• Stomach acid denatures dietary protein for digestion by pepsin, trypsin, and chymotrypsin

Analysis of Protein Structure

• Analysis of primary structure (sequence analysis) reveals effects of genetic mutations and shows homologies within protein families
• Three major steps:
  • Amino acid composition: Quantitative analysis after protein digestion by acid hydrolysis
  • Fragment sequencing: Specific hydrolysis by chemicals or proteolytic enzymes, followed by Edman degradation of each fragment
    • Trypsin hydrolyzes the carbonyl side of lys and arg
    • Chymotrypsin hydrolyzes the carbonyl side of aromatic rings (phe, tyr, trp)
    • Cyanogen bromide cleaves on the carbonyl side of methionine
    • Edman degradation: Sequential removal and identification of N-terminal amino acids using phenylisothiocyanate, done for peptide sequences up to 50-60 residues long
  • Fragment linking: Cleavage at different points yields overlapping fragments, which helps deduce the overall primary structure
    • Disulfide bonds can be cleaved by reaction with reducing agents like mercaptoethanol

Methods for Studying Higher Order Structure

• Methods for purification and characterization of proteins take advantage of tertiary and quaternary structure
• Protein precipitation increases salt concentration, dehydrating proteins and causing aggregation and precipitation at unique salting-out points
• Proteins can then be separated based on differential solubility

Chromatography

• Chromatography separates proteins by their different interactions with a stationary matrix
  • Gel exclusion: Porous matrix excludes molecules above a certain molecular weight
  • Ion exchange: Charged matrix binds proteins of opposite charge, removed with a salt or pH gradient
  • Affinity: Ligands on the matrix bind specific proteins, eluted with unbound ligand or pH gradients
  • Reverse-phase: Nonpolar matrix adsorbs molecules, desorbed by increasing nonpolar solvent concentration
  • High-performance liquid chromatography: Microparticulate resins require high pressure, yielding higher resolution separations
• Centrifugation separates proteins based on sedimentation rate (size and shape), measured in Svedberg units (S)

Dialysis

• Dialysis places a protein sample in a semipermeable membrane bag, immersed in buffer solution
• Smaller molecules diffuse through the membrane, larger molecules are retained
• Dialysis removes salt or reversibly bound cofactors or inhibitors, using semipermeable filters with specific molecular weight cut-offs

Electrophoresis

• Electrophoresis applies protein samples to a porous gel or cellulose acetate strip in buffer solution
• Proteins migrate to oppositely charged electrodes at rates depending on size, shape, and charge
• Electrophoresis purifies and characterizes proteins

Fingerprinting

• Protein fingerprinting produces a distinct and specific pattern
• When proteins are subjected to selective proteolytic digestion and separated in two dimensions with chromatography and electrophoresis

X-Ray Diffraction

• X-ray diffraction produces electron density maps from crystal lattices of proteins
• Diffraction patterns are then converted to 3-D pictures of the protein’s structure

Nuclear Magnetic Resonance (NMR)

• NMR derives a 3-D image of proteins in solution by measuring resonance frequencies of atomic nuclei
• This method provides information on the solution structure of a protein, but requires known primary protein structure

Key Points About Analysis of Protein Structure

• Tertiary structure is the complete 3-D structure of a protein, with hydrophobic side chains inside and hydrophilic side chains outside
• Quaternary structure describes the subunit composition of a functional protein
• Weak chemical bonds stabilize higher orders of protein structure, allowing conformational changes
• Disulfide bonds further stabilize secreted proteins
• Breaking weak chemical bonds results in a loss of higher-order structure and function

Model Proteins: Hemoglobin and Myoglobin

• Hemoglobin and myoglobin are both well-studied and clinically relevant
• Help illustrate basic principles of protein structure and function
• Hemoglobin transports O2 from the lungs to the tissues and is found only in erythrocytes
• Myoglobin stores O2 only in skeletal muscle
• Table 3-1 compares the characteristics of hemoglobin and myoglobin
  • Hemoglobin transports O2 and is only located in the erythrocyte
  • Myoglobin stores O2 and is only located in skeletal muscle
  • Hemoglobin has low O2 affinity in tissue
  • Myoglobin has high O2 affinity in tissue
  • Hemoglobin changes its O2 affinity with PO2
  • Myoglobin does not change its O2 affinity with PO2
  • Hemoglobin is allosteric
  • Myoglobin is not allosteric
  • Hemoglobin is a tetramer
  • Myoglobin is a monomer
• Hemoglobin binds oxygen reversibly, becoming saturated with oxygen at high oxygen concentration in the lungs
• Oxygen is released at lower oxygen tension where it is used for aerobic metabolism
• Myoglobin binds oxygen more tightly than hemoglobin and serves as an oxygen buffer, releasing oxygen when tissues become hypoxic
• Unlike hemoglobin, myoglobin does not change its affinity for O2 as it binds increasing molecules of O2
• Specific monomer units that make up hemoglobin depend on the developmental stage of the individual
• Hemoglobin F, composed of α- and γ-chains, is the predominant form in the fetus
• Hemoglobin A, composed of α- and β-chains, is the predominant form in adults

Hemoglobin

• One subclass, hemoglobin A1c (HbA1c), is formed from a spontaneous reaction between blood glucose and the amino-terminal valine residue of the β-globin chain
• HbA1c increases in patients with elevated blood glucose
• Myoglobin is always in monomeric form and has no quaternary structure
• Hemoglobin and myoglobin have similar tertiary structures:
  • All are α-helical with connecting regions between helices
  • Both are compact with hydrophilic residues toward the outside and hydrophobic residues toward the inside
  • Both have a hydrophobic pocket for one heme prosthetic group
• Structure determined by hydrophobic and hydrogen-bonding interactions between amino acid residues
• In myoglobin, surface amino acids are primarily polar, promoting protein solubility

Heme Structure and Function

• Heme is a planar iron-containing porphyrin ring with iron held in the center by coordination bonds
• Four coordination bonds are to pyrrole nitrogens, a fifth to the proximal histidine (His-F8), and the sixth to O2
• Although the heme iron binds O2, it is not oxidized and remains in the Fe++ form
• Methemoglobin is where the iron has been oxidized to Fe+++ and can no longer bind O2

Cooperativity

• Cooperativity is where the binding of a ligand to one monomer of a multimeric protein affects the binding of that ligand to an adjacent monomer
• Hemoglobin demonstrates this

Allosterism and Hemoglobin

• Allosterism describes the change in affinity for binding caused by the binding of another ligand (allosteric = other site)
• Allosterism is not the same as cooperativity
• Cooperativity creates the sigmoid curve
• Allosterism shifts the curve to the right or left, and affects cooperativity
• Higher affinities (positive allosteric effect) shift curves to the left, lower affinities (negative allosteric effect) shift curves to the right
• Allosteric effectors for hemoglobin shift the curve to the right, decreasing affinity (negative allosteric effect)

-2,3-Bisphosphoglycerate (2,3-BPG)

• 2,3-Bisphosphoglycerate is a metabolite that is present in high concentrations in RBCs and is the principal allosteric effector for hemoglobin
• One BPG molecule binds reversibly to a tetramer with the monomers all in the T-form and stabilizes the T-form
• 2,3-BPG has little effect on the binding of oxygen to hemoglobin at high P02, but promotes release of O2 from hemoglobin at low P02

Carbon Dioxide

• Reaction of CO2 with N-terminal amino groups of globin polypeptides forms carbamate: CO2 + Hb-NH3+ → Hb-NH-COO-
• In this form, hemoglobin transports about 15% of the CO2 carried in blood
• Carbamate formation favors salt bridge formation and lowers the O2 affinity of hemoglobin

Protons

• The Bohr effect is the loss of affinity for O2 with decreasing pH
• Protons shift the equilibrium toward the T-form by binding to surface amino acids
• Through equilibrium with protons, hemoglobin also contributes significantly to the buffering capacity of the blood
• Increased body temperature reduces hemoglobin O2 affinity, allowing increased unloading of O2

Carbon Monoxide Poisoning

• This stabilizes the R-form of hemoglobin
• Binds with a 200-fold greater affinity
• This shifts the subunit into the R-form, facilitating the loading of the molecule with Oxygen

Fetal Hemoglobin

• 2,3-BPG has weaker binding to fetal hemoglobin (HbF)
• Reduced negative allosteric effect of 2,3-BPG, leading to a small increase in O2 affinity of HbF relative to HbA

Hemoglobinopathies

• Genetic diseases caused by structural alterations in the globin chains or by altered rates of globin synthesis

Structural Alterations in Hemoglobin

• Sickle cell hemoglobin (HbS) is caused by a mutation (β6 Glu → Val)
• This leads to the formation of linear polymers of deoxygenated HbS
• Hemoglobin Boston is caused by a tyrosine substitution

Altered Rates of Globin Synthesis

• Unbalanced production of either a-globin or β-globin leads to thalassemias
• aprogressive loss of a-globin genes results in more severe anemias, which affect the fetus

Key Points About Model Proteins

• Hemoglobin structure increases its affinity for oxygen in the lungs and decreases its affinity for O2 in the tissues
• Myoglobin has no quaternary structure
• The different quaternary structures for hemoglobin throughout development reflect specialized needs for O2 transport
• Hemoglobin changes its affinity for O2 as it binds oxygen
• Allosterism results from binding a ligand, or effector, at a site other than the primary site
• Hemoglobinopathies are diseases caused by alteration either in hemoglobin structure or in its rate of synthesis