Ch2
What are the properties of the bonds and interactions between and in Water molecules?
-Water is polar because of electronegativity difference
-Covalent O-H bonds
-Water hydrogen bonds with itself causing increased Boiling point, melting point, and specific heat, heat of vaporization (physical properties of water), Cohesion and adhesion as well as dissolving polar molecules
-also contains Dipole-dipole interactions, LDF, allows for hydrophobic affect (non-polar molecules reduce H-bonding amount).
Amino acids have amino group, carboxyl acid group, hydrogen atom, R group.
-At neutral pH COO- is deprotonated and NH3 is protonated
-R group adds variation. Nonpolar, aromatic, polar, neutral, charged
Hydrophobic residues are inside protein, hydrophilic are outside bc of interaction with H2O
- Charged amino Acids form salt bridges, Cysteine makes disulfide bonds, Proline is Kinky and Glycine is hyperflexible
All bonding interactions in protein structure
Hydrophobic interactions (strongest)- non-polar side chains cluster inside protein away from water (releasing ordered water increasing entropy)
H-bonds- Forms between peptide backbone and side chains to stabilize secondary, tertiary and quaternary structure
Ionic interactions (salt bridge)- attraction between oppositely charged AA (lys, Glu) (Arg, Asp)
Van Der Waals- atoms have small attractive interactions
Disulfide bonds- strong covalent bonds of 2 oxidized cysteine molecules to stabilize tertiary proteins
Peptide bonds- covalent bonds formed between AA in primary sequence
Folding is due to hydrophobic affect everything else is to stabilize
Why does O2 bind to Hemoglobin cooperatively. Include Concerted model and sequential model.
Hemoglobin has 4 subunits. When 1 molecule O2 binds it changes Hemoglobins affinity for more O2.
-Hemoglobin with no O2 bound favors the T state which has low affinity for O2. When O2 binds a conformational shift happens moving Fe2+ slightly into the plane of Heme. This shifts favorability toward R state that has higher affinity for O2. As O2 binds affinity changes exponentially causing sigmoidal shape.
How do enzymes lower activation energy in order to increase chemical reaction rate?
1) Enzymes bring reacting molecules closer to each other and in the right position to react
2) Enzymes bind and stabilize the transition state
3) Enzymes binding energy (energy from noncovalent ES interaction) to overcome energy requirements
4) Enzymes provide AA for acid-base catalysis and can form temporary covalent bonds to lower Ea.
5) Provide a favorable environment (polarity, charge) to favor reaction
What is the significance of H-bonds in macromolecules?
help stabilize proteins (secondary, tertiary, quaternary), Nucleic acids (AT & CG), Carbohydrates (-OH allowing glucose to be soluble), and lipids (head group)
Approximate net charge of a peptide, given pH and pKa values + Peptide sequence
ex: MANISHMISHRA
at pH = 1, 7, 13
@pH=1 it is +4
@pH=7 it is +1
@pH=13 it is -1
as pH increases the protein becomes more deprotonated
-When pH=pKa 50% of group is protonated 50% deprotonated
What does Vitamin C do for post translational modification of AA side chains and its impact human health & disease
Vitamin C is a cofactor. In collagen synthesis is helps with hydroxylation of Lys and Pro. Hydroxylated Proline is needed for stabilizing Collagen triple helix and hydroxylysine is needed for collagen cross-linking.
Vitamin C deficiency leads to compromised collagen, degeneration of connective tissue called scurvy.
Concerted model (MWC)- says that all 4 Hemoglobin subunits are either T state or R state. All subunits change together rather than separately.
How do small molecules inhibit enzyme activity?
Molecules can bind to regions of the enzyme and impact its function. This binding impacts enzyme structure and that determines FUNCTION.
Competitive inhibition- Inhibitor binds at active site so substrate cannot bind there. Causes an increase in Km because more substrate is required to achieve 1/2 Vmax. Circumvented by adding more substrate
Noncompetitive inhibition- Inhibitor binds at allosteric site causing conformational change of enzyme making catalysis less effective. This causes a decrease in Vmax.
Uncompetitive inhibition- Inhibitor binds to ES complex preventing reaction from completing. This decreases Km as substrate cannot dissociate from enzyme and decreases Vmax because total free enzyme decreases.
Mixed inhibition- Can bind at allosteric site or ES complex. Vmax decreases, but Km can increase or decrease depending on whether inhibitor favors allosteric site or ES complex.
Irreversible inhibitors- Forms bond with enzyme permanently preventing it from binding to substrate ex: suicide inhibitors
pH is -log of [H+] high [H+] means low pH
pKa is the -log(Ka) a High Ka means a low pKa and indicates a stronger acid
Kd is the dissociation constant Ka is P/r and Kd is 1/Ka so Kd is R/P
low Kd is high affinity
What are the primary, secondary, tertiary, and quaternary structures of proteins?
Primary- AA sequence connected by peptide bonds (covalent) ex: ATGVIL
Secondary structure- arrangement of amino acids with respect to the polypeptide backbone. ex: a-helix, B-sheets, B-turns, B-loops, random coils
Tertiary- 3D shape of chain due to interactions between side chain. Stabilized by Hydrophobic affect, Hydrogen bonds, ionic interactions (salt bridges), disulfide bonds, and Van der Waals.
Quaternary- Arrangement of 2+ polypeptide subunits into a protein complex. ex: hemoglobin
Explain the regulation of protein homeostasis and how defects in protein homeostasis relate to human disease
Proteostasis- continuous maintenance of proteins
Structure determines function. Lose structure loss function
Molecular chaperones assist proteins in folding to prevent misfolding
Ubiquitin-proteosome system- labels and degrades misfolded/damaged protein
Autophagy/lysosome- big proteins fuse with lysosome and are degraded
UPR- increase chaperone synthesis and promote degradation
defects in proteostasis result in aggregation of misfolded proteins causing disease.
Accumulation of B-amyloid: Alzheimer
Accumulation of a-synuclein: Parkinsons
Accumulation of huntingtin: Huntington disease
Mutation of CFTR gene causes misfolded protein
Misfolded proteins in brain aggregate causing Prion disease
What is sequential model?
Sequential model- Binding of O2 changes 1 subunit into R state influencing other subunits to change as well.
3 mechanisms for how enzymes are regulated in cells
1) Allosteric regulation- Bind to allosteric site of enzymes to regulate them. Can be enzymes product (homotropic) or another molecule (heterotropic)
2) Covalent modification- Adding/removing phosphate groups to active/inhibit enzymes. ex: Acetylation, methylation
3) Cleavage of enzyme precursor- Zymogens are proteins with extra AA sequence and inactive. Cleave part of protein to activate enzyme.
What is Henderson-Hasselbalch?
pH = pKa + log([A-]/[HA])
can calculate ratio of conjugate base to weak acid needed in a buffer
ex: pH =7.4 and pKa = 6.4 the 1=log(A/HA) then 10 = [A]/[HA]
Therefore, A must be 10x more abundant that HA. If A is 20mM then Ha must be 2mM for a total of 22mM buffer
When [A]=[HA] then pH=pKa
What are the protein separation techniques?
Goal to separate proteins from one another
separate by size, charge, binding, solubility
Size exclusion Chromatography- Large elute first (small trapped in pores)
Ion-exchange Chromatography- separate by charge
Affinity Chromatography- binding affinity
HPLC- uses high pressure to move proteins and improve resolution (separation)
Isoelectric Focusing- has pH gradient that allows protein to travel down. Proteins travels until it reaches its pI (becomes neutral) and stops.
Gel electrophoresis (Native)- small proteins elute first (not contribute to purifying). Visualize and analyze components.
SDS PAGE gel electrophoresis- detergent that adds (-) charge for size separation only.
Reducing SDS PAGE- breaks disulfide linkages.
2D Gel electrophoresis- separates complex protein mixtures. Combines IEF and SDS page. (separate with pI and size)
Techniques for determining Protein structure*
X-ray Crystallography- 3D structure of proteins. Crystalize protein, then use X-rays. Study active sites and makeup of protein
NMR- study proteins in solution. Use magnetic field to radiate nuclei. Observe dynamic "breathing" proteins. Structure + dynamics
NOESY (type of NMR)- tells u distance between nuclei in 3D space
TOCSY (type of NMR)- tells u about nuclear spins of nuclei connected by covalent bonds.
Cry-EM- Freeze protein. Traps protein in its native conformation them analyze in electron microscope. Use computer to reconstruct the particles into 3D structure. Useful for LARGE COMPLEX PROTEINS
Antibody binding to understand protein structure and function
Abs have Y-shaped structure made of light and heavy chains. The 2 tips at the top of the Y are variable regions that are made for a specific Ag (rest are constant region).
Binding site (variable region) recognizes epitope on Ag and binds noncovalently. Changes in the STRUCTURE (variable region) of Abs affect their FUNCTION (Ags they bind to). Can also have induced fit
If a plane crashes on the border of 2 countries where do you bury the survivors?
You don't bury Survivors :)
What is the significance of buffers?
They resist changes in pH when acids or bases are added. Many processes require a specific pH range
ex: bicarbonate buffer keeps blood around 7.4
Phosphate buffer works in cytosol oof cells
Protein buffer works by using ionizable AA to bind to access H+ to stabilize pH in cells and blood. Hemoglobin does this as well especially in blood
What are protein sequencing techniques?
Edman degradation- identify AA one at a time starting from N-terminus (for short peptides)
Mass spectrometry (MS)- Measure protein mass (short AA sequence)
MALDI MS (ionization method)- uses light to ionize proteins (move to gas phase) and deduce m/z (if you know charge can deduce mass). Good for deducing m/z of purified proteins
ESI MS (ionization method)- Spray liquid sample through a charged needle (turns into gas). Produces many charged states of same molecule. Good for liquid samples and small molecules
Tandem MS/MS- Break ion into smaller pieces to determine AA sequence. Gives u structural information besides mass.
LC-MS/MS - separates molecules then release them 1 by 1 to measure m/z then fragments after to detect amino acid sequence. Good for complex mixtures. Separate then deduce m/z and identify compounds
Fetty WAP boi!
Muscle Contraction to understand protein structure and function
Myosin heads have ATP binding site and actin-binding region. Actin makes filaments with myosin head.
-Ca2+ levels rise in cell, Ca2+ binds to troponin, tropomyosin moves away from myosin-binding site on actin, Myosin head can now bind to actin. ATP is hydrolyzed and myosin pulls the actin filament. ATP binding to myosin causes myosin to release actin. Protein FUNCTION is dependent on the STRUCTURE of myosin-actin.
Name 3 other songs by the person who created Trap queen
679
My Way
Again
Birthday