Week 1 - Quiz 1 Flashcards
Physiology
The study of the function and integrative process of Life (all levels of structural complexity)
Frames function in evolutionary, environmental, ecological and behavioral context.
Aims to achieve translation of knowledge, through a cross-disciplinary approach, into human health.
Who was the father of modern physiology?
Claude Bernard
Who is Claude Bernard?
Father of modern physiology. Laid the foundation of physiology as an empirical, experimental science.
Proteins
Linear biopolymers composed of 20+ different aa’s linked by PEPTIDE BONDS
Primary AA structure
the unique sequence of aa’s for each protein
Biological function of proteins depends on
3D structure - tertiary structure
Secondary AA structure
primary structures spontaneously fold (apolar aa in core, and polar aa’s on surface). Involves H-bonds between peptide bond carbonyl groups and peptide bond amide hydrogens. Form alpha helix, parallel beta pleated sheets, and anti-parallel beta pleated sheets.
What formation comes after secondary aa structures?
Super-secondary structures, then domains, and tertiary structures.
Quaternary structure
if the protein is comprised of multiple subunits, the association of subunits forms the quaternary structure
Enzymes vs structural proteins
Most enzymes are soluble globular proteins - spherical or ellipsoidal in shape.
Structural proteins are elongated with unusual shapes and very stable structures.
Name a few important structural proteins
alpha-Keratin, Collagen, and Elastin are important extracellular structural proteins
Involve UNUSUAL aa’s (hydroxyproline) and sequences. Or they have unusual crosslinks that allow the protein to stretch without breaking.
What kind of bond holds aa’s together in a protein
PEPTIDE bond between the carboxyl group of one aa and the amino group of the other.
Where are proteins synthesized
on ribosomes from N-terminal to C-terminal
What 2 features of a peptide bond limit the number of conformational states possible for a polypeptide?
The strong electronegativity of the carbonyl O compared to the amide N causes the peptide bond to delocalize electrons, creating 2 major resonance arms. O-C=N form accounts for 40% of the structure, while the O=C-N accounts for 60%
Are peptide bonds freely rotating?
Because of the resonance, and since double bonds are always planar, peptide bonds have planar character with little bond rotation around the amide bond.
How are R groups aligned in two adjacent aa’s?
Preferred orientation is trans, minimizing steric hindrance. This limits the number of available conformational states.
What is the exception to the common “trans” preference of R groups?
Proline: cyclic side chain
In an AA chain, what are the 3 linkages/angles?
Peptide Bond: C-N: rigid and trans
Phi: N-Calpha (angle)
Psi: Calpha-C (angle)
Phi and Psi are free to swivel, limited only by steric hindrance between atoms of the polypeptide chain and R groups
What is known about spontaneous protein folding?
- Hydrophobic forces have a large role in driving folding
- Folding begins with local interactions that lead to a secondary structure. Then, close packing of hydrophobic groups excludes water - forming a DOMAIN (basic unit of tertiary structure.
- Correct and incorrect folding occurs REVERSIBLY. Correct interactions cause alignments that facilitate additional correct interactions. This increases the stability of that folding pattern.
- the most THERMODYNAMICALLY stable formation is attained due to this incremental way of folding.
What does the free-energy funnel represent?
As incremental protein folding proceeds, there is a decrease in the number of conformations present. With rare exceptions, this narrowing represents the NATIVE CONFORMATION of the protein, and the one that is BIOLOGICALLY ACTIVE.
Alpha helixes are (left/right) handed?
RIGHT
In Alpha helixes, how many residues are there per turn?
3.6 residues per turn - 5.4 angstroms (Between R groups that stick out)
What type of bond holds together alpha helixes? And where are they located?
H+ Bonds:
Between carbonyl of AA (i) and amide proton of AA (i+4)
What are Hydrogen bonds?
Weak bonds due to the electrostatic attraction between the proton (on the amine group) and the electronegative carbonyl O
How to R side chains project in alpha helixes?
Radially outward from the helix axis, minimizing steric repulsion
How are H+ bonds aligned in an alpha helix?
Parallel to the helix axis.
What characteristic do alpha helices on the surface of a protein have?
AMPHIPATHIC: hydrophilic face projecting out to the solvent, and hydrophobic face projecting in to the apolar core of the protein
How are beta pleated sheets stabilized?
Hydrogen bonds BETWEEN carbonyl O and amide H of NON-CONTIGUOUS parts of the polypeptide chain.
How are H+ bonds aligned in beta sheets?
Co-planar. In plane with the sheet.
How are R side chains aligned within beta sheets?
Alternately project above and below the plane of the sheet
Which part of a protein to beta sheets usually form?
the hydrophobic core or proteins.
Parallel vs Antiparallel Beta Pleated Sheets?
Parallel: peptides are aligned in the same direction
Antiparallel: Peptides alternate in direction
Can parallel and antiparallel strands exist together?
Yes, mixed beta sheets - All are EQUALLY stable and allow dense packing of aa’s in a proteins core.
How is it possible to antiparallel beta sheets to be a part of the same peptide chain?
require a U-turn: Reverse turn, Beta-turn, or Hairpin turn
What is the purpose of a secondary protein structure?
Solves the problem of placing peptide bonds (polar) into hydrophobic (apolar) interior of a globular protein. O and NH atoms are able to hydrogen bond with each other rather than with water.
What AA affects secondary structures significantly?
Proline. Ring structure prevents it from forming a Hydrogen bond required for alpha helix/beta sheet.
Proline residues are often found where alpha helixes or beta sheets end. This is one way primary sequence directs folding.
The possible conformations of the peptide backbone are limited by what two things?
- The fact that peptide bonds (double bond characteristics) are more stable when planar and trans
- Rotation around the other bonds of the peptide backbone (phi and psi angles) are limited.
What is the Ramachandran Plot?
Many combinations of phi and psi angles cause the side chains of AA’s to clash (unfavored). Ramachandran Plot: A plot which illustrates favored combinations of phi and psi angles.
What is a super-secondary structure
A specific grouping of secondary structural elements. Secondary structures making stable associations with one another (folding on top of another)
What is a domain?
As parts of the peptide chain form associations and supersecodary structures, domains are achieved. STABLE!
Domains are the smallest thermodynamically stable units of protein structure. Often account for a particular part of an enzyme’s active site. OR may catalyze one part of a complex reaction sequence.
Tertiary structure
term that describes the final thermodynamically stable structure of a single polypeptide chain. (if there is only one domain, then THAT is the tertiary structure)
What are metamorphic proteins
A protein that can exist as 2 different structures at approximately equal energy - in equilibrium. Thermodynamic folding funnel with two wells of similar depth instead of one.
Name 3 fibrous proteins
alpha-Keratin, Collagen, and Elastin
Alpha Keratin characteristics:
Primary component in hair and nails.
Consists of two right handed alpha helices - that are intertwined in a left-handed super-coil (alpha coiled coil)
What is an alpha coiled coil? And what kinds of bonds hold it together?
In Keratin: A super-coil consisting of two right handed alpha helices that are intertwined in a left-handed supercoil.
Bonding between coils is both non-covalent and covalent.
Non-covalent: hydrophobic interactions, ionic bonds, H-bonds
Covalent: Disulfide bonds between adjacent cysteine residues
What characteristics do disulfide bonds present? (Moderate amounts and Large amounts)
Moderate number of disulfide bonds: Allows hair to stretch but then return to its original shape
High number of disulfide bonds: Give nails, horns, claws, and hooves their rigid character
What does coiled coil proteins include?
include intermediate filaments that provide internal scaffolding for cells, myosin, and tropomyosin (involved in muscle action)
What is the most abundant protein in the body? and at what percent?
Collagen - 20-25% of total protein
Collagen characteristic:
Collagen exists as a superfamily of molecules
typically, is a long, rigid structure in which 3 polypeptides are wound around each other in a rope-like triple helix
What are individual collagen chains called and which direction do they turn?
alpha chains
Left handed helices (NOT left handed alpha-helices)
How is the collagen triple helix aranged
Each alpha chain is a left handed helix. Three of them come together to form a right-handed triple helix (Coiled coil or super-helix)
What are collagen microfibrils
When multiple collagen triple helices interdigitate, they form microfibrils
Which amino acids are very prevalent in Collagen?
Proline: proline rings stack along the outside of the helix, giving it rigidity
Glycine: found at every third position of the polypeptide chain, so it can fit into the restricted central space where the three strands come together.
What repeating sequence does collagen have?
Gly-X-Y-
X is frequently proline
Y is often either hydroxyproline or hydroxylysine
Why does collagen aggregate into long fibers?
The elongated triple helix places many amino acid side chains on the surface, allowing bond formation between neighboring chains - creating aggregations.
How are Hydroxyproline and Hydroxylysine formed? When? and Why?
How: post-translational hydroxylation reactions or proline and lysine
When: After they’ve been incorporated into the polypeptide chain
Why: Serve to stabilize the triple-helical structure
What does ascorbate have to do with collagen?
The enzyme that catalyzes the hydroxylation of hydroxyproline and hydroxylysine requires ascorbate (vitamin C).
What happens when there is an ascorbate deficiency?
Without ascorbate, the enzyme cannot catalyze the hydroxylation of lysine and proline. This deficiency results in scurvy. Symptoms of scurvy (bleeding gums, easy bruising, cork screw hair) is a result of the decreased tensile strength of collagen.
What is another way covalent crosslinks are formed in collagen?
Lysyl Oxidase converts some lysine into allysine (CH=O on the end of a lysine) - allowing it to undergo bond formation with neighbors.
Draw/List the biosynthesis of Type 1 Collagen
Synthesis, post-translational modification, and triple helix assembly form inside the cell - forming a Procollagen. The pro collagen molecule is secreted into the extracellular matrix. Here peptidases cleave the N and C terminal pro-peptides - forming a mature collagen triple helix (Tropocollagen). The mature tropocollagen self assembles into fibrils, and subsequently cross-links to form mature collagen fibers.
Properties of Elastin
Connective tissue protein with rubber-like properties
Where is elastin found
lungs, large arterial walls, elastic ligaments
What type of aa is the elastin polypeptide comprised mostly of?
Small, non-polar amino acids (glycine, alanine, valine) - also rich in proline and lysine, but contains few hydroxyl derivatives of those.
What is the precursor for elastin? Where is the precursor made?
Tropoelastin. Secreted into the extracellular space, where it interacts with specific glycoprotein microfibrils.
What special structure does elastin contain?
Desmosine: a structure made by cross linking lysine and allysine residues.
How is desmosine made?
Some Lysyl side chains are oxidized to form allysine. Allysine cross-links with lysine amino group of neighboring polypeptides to produce elastin. The extensively interconnected nature produces the highly elastic properties.
What is alpha1-antitrypsin (a-AT)
a small protein that inhibits several proteolytic enzymes in bodily function.
Critical function: inhibits elastase (break pedal)
What is elastase?
An enzyme (proteolase family - breaks down enzymes) which breaks down elastin. Elastase is released by neutrophils. Can destroy the alveolar epithelium if unregulated (by a-AT).
What can insufficient functional a-Antitrypsin cause?
Emphysema (alveolar epithelium breaks down)
Genetic Causes for insufficient functional alpha-antitrypsin
alpha-AT deficiency: predominancy due to inheritance of two mutant alleles (Z and S). The Z allele causes a more severe deficiency and is due to a E342K (wild type) substitution causing alpha-AT to be retained inside the cell.
Environmental causes for alpha-antitrypsin deficiency
Alpha-AT contains a methionine residue (making it essential for binding target proteins - like elastase). Elements in cigarette smoke cause oxidation of the Met residue, making alpha-AT unable to bind - and inactivate elastase) methionine - to - methionine sulfide (O=S)
What is the benefit of coopertivity
increases ability of Hb to release O2 in tissues
Right/Left shift means what in terms of O2 affinity?
Right = Lower affinity (more likely to release O2 to tissues) Left = Higher affinity (more likely to bind O2)
What does 2,3 DPG or BPG do
Allows Hb to adapt to hypoxia - more easily releasing O2
What happens in Sickle cell disease?
Inherited mutation of the beta globulin gene, causing a change from Glu (E) to Val (V) at position 6.
How does Val in position 6 effect the globulin?
This allows DeoxyHbS to polymerize into microfibrils that distort the RBC shape. Causes both hemolysis and vasooclusive disease
How does the globulins conformation change with/without O2 binding?
Without O2: His residue of the Hb polypeptide chain is able to pull Fe (heme) away from the plane of the porphyrin ring.
When O2 found: O2 binding pulls the Fe back into the plane of the ring. Moves the His residue and its whole section of the polypeptide chain.
What is an allosteric regulator?
Allosteric regulator or Effector:
molecule that binds to a protein and induces a conformational change with alters the affinity for a substrate at some other site.
Draw out the equilibrium equation that drives O2
DPG-HHb-CO2 + O2 HbO2 + CO2 + DPG
when O2 is high, equilibrium shifts to the right, and O2 binds to Hb
When Co2 or DPG are high, equilibrium shifts to the left and O2 dissociates from Hb.
What are the pH, pO2, pCO2, and HCO3 in arterial blood, typically?
pH: 7.35/7.45
pO2=80-100mm Hg
pCO2= 35-45 mm Hg
HCO3- = 22-26 mM
Define Allosteric Enzyme
enzyme that has more than one binding site
Positive vs Negative Effectors
Effectors or allosteric regulators: modify allosteric enzymes. Bind non-covalently and change the affinity of the enzyme for substrate.
Positive: increase the affinity of the enzyme
Negative: effectors that inhibit the enzyme
Homotrophic vs Heterotrophic Effectors
Homotropic Effectors: Substrates that also act as an effector
Heterotrophic Effectors: An effector other than the substrate
What type of effectors are H+, CO2, and DPG?
Heterotropic negative allosteric effectors. Decrease the affinity of Hb for O2.
What type of effector is O2?
Positive homotropic allosteric effector of O2 binding.
It is a negative allosteric effector of H and CO2 bind.
What is the Bohr effect and Isohydric shift?
the reciprocal relationship between O2 and H binding. Lowered affinity of Hg for O2 when there is an increased concentration of H+ due to the increased concentration of CO2 in the blood.
Describe how pKa effects the Bohr effect?
Conformational changes in the hemoglobin molecule create microenvironmental effects which shift the pKa of specific residues (mostly histidine). This change also effects and changes the H binding.
In dyoxyHb, Asp is near His. The negative C terminus of the Asp is near the N in the His ring, therefor it is energetically favorable for the N to be protonated (NH’s pKa is higher, meaning more protonated at a given pH). Having the His protonated makes it favorable for the Asp to stay near it, increasing the stability of deoxyHb (T-state).
When changing to OxyHb (R state) His and Asp are moved apart, therefor the pKa drops, and the H+ comes off.
Where does 2,3 DBG bind?
Binds to a specific site in a central cavity between the beta subunits. Binding is by ionic interactions. Lots of negative charges on DPG allow it to bind to positively charged groups (8) of Hb beta.
DPG changes occur over days as the body is adapting. Unlike CO2, H+, DPG levels don’t change instantly as blood travels throughout the body.
How does DPG effect O2 binding curves?
1) Without any DPG, Hb would be more like myoglobin and nearly useless for delivering O2 from lungs to tissue. DPG in normal blood = 5mmol/L. DPG stabilizes the T-State, making it easier for Hb to release O2.
2) DPG levels increase at high altitudes. With less O2 at high altitudes, tissue becomes hypoxic. By increasing DPG conc. RBC adapt to hypoxia, making it easier for O2 to dissociate from Hb and go to tissues. Other causes of tissue hypoxia (anemia and smoking) cause an increase in DPG as well.
What is Hemoglobin Milwaukee (HbM)
Rare, dominant mutation of the beta subunit (E67V). Changes allow the Fe to be more easily oxidized. Affected pos have high levels of MetHb - few consequences in every day life.
Sickle cell disease (anemia)
Homozygous recessive disease - most common in hemoglobinopathy.
Where is the mutation and what kind - in SCD
Point mutation E6V. Glu is hydrophilic (good bc its on the surface of the beta chain). Valine is hydrophobic - creating a sticky patch on deoxyHb leading to polymerization of Hb tetramers into long chains. Fibers cause sickle shape and reduce deformability, causing difficult with passage through microcirculation. Also: Shorter erythrocyte half-life and chronic hemolytic anemia.
Results in its having mainly HbS (2 normal adult alpha globes, and 2 sickle adult beta globins).
What interaction stabilizes sickle cells?
A critical contact made by beta 6 Val of one molecule and a hydrophobic acceptor (formed by Leu and Phe surrounded by hydrophilic residues) of another molecules’ beta subunit. The negative charge on Glu prevents this from happening normally.
What are the 3 variables that effect the rate and extend of polymer formation in SS RBC’s?
3 INDEPENTEND variables:
1) Degree of Deoxygenation. Deoxygenated HbS forms insoluble polymers.
2) Intracellular hemoglobin concentration
3) Relative amount of HbF present. Hbf INHIBITS polymerizations.
What other abnormalities does the generalized deformation of the HbS erythrocyte lead to?
- Dysregulation of red cell volume:
Sickling -> Membrane distortion -> Ca2+ leakage.
The Gardos Effect: Increased intracellular Ca2+ activates the Gardos change (export of K). Loss of K, Cl, and H20 = dehydration which increases HbS concentration. - Adhesion to the microvascular endothelium: increased surface exposure of adhesive components
What 2 drug therapies for SCD?
Hydroxyurea: Antitumor drug. Stimulates HbF production. More HbF = less HbS
L-Glutamine oral powder (Endari) approved July 2017. Works as an anti-oxidant.
What are Thalassemias and what are 2 types?
Thalassemias are an imbalance in the concentration of alpha or beta globin chains.
Alpha - Thalassemia: Decrease in alpha-globin chain: Redundancy provided by four alpha genes means its rare.
Beta: decrease in beta-globin chains. Mutations in the adult beta globin gene can affect chain termination, transcription activation, and RNA processing. Can get promoter mutations, frameshift mutations, splicing mutations
Hemoglobin C
A point mutation in codon 6 of the adult beta-globin makes a Glu to Lys substitution. 2 normal alpha subunits and 2 abnormal beta subunits causes mild hemolytic anemia. HbC allele is most commonly found in the compound heterozygous genotype with HbS allele. HbSC is a less severe for of SCD.
HbA1c
most abundant form of glycosylated hemoglobin (blood glucose attached to a lysine residue in Hb). Normally present in low acts, but become high in diabetics.
