BIOC12: BIOCHEM (Midterm) Flashcards
what are the 4 groups of macromolecules?
- nucleic acids
- proteins
- carbohydrates
- lipids
a group of molecules that are largely hydrophobic and only sparingly soluble in water
lipids
what does in vitro mean?
outside a living cell
protein or RNA biomolecules that function as reaction catalysts to increase the rates of biochemical rxns
enzyme
sequences of biochemical reactions coordinated and controlled by a cell in response to available energy
metabolic pathway(s)
mechanisms that facilitate communication b/w cells, often initiated through the binding of small molecules to proteins called receptors
signal transduction
nitrogen-containing molecules that function primarily as the building blocks for proteins
amino acids
what are nucleotides made of?
- a nitrogenous base
- a 5-membered sugar (ribose / deoxyribose)
- 1-3 phosphate groups
what are the 5 nucleotide bases?
adenine
guanine
cytosine
thymine
uracil
compounds formed only of carbon, oxygen, and hydrogen (with a 2:1 ratio of hydrogen to oxygen atoms)
simple sugars (aka carbohydrates)
when polar and non-polar chemical properties are contained within the same molecule
amphipathic
a molecule consisting of a polar carboxyl group (COOH) covalently linked to a non-polar extended hydrocarbon chain
fatty acid
a covalent bond b/w the alpha amino group of one a.a. and the alpha carboxyl group of another a.a.
peptide bond
any of a group of small biomolecules that serve as reactants and products in biochemical reactions within cells
metabolite
the rate at which reactants and products are interconverted in a metabolic pathway
metabolic flux
metabolite/hormone/peptide that binds to target proteins and alters their structure and function to control biochemical processes
ligand
a set of metabolic processes and reactions that uses oxygen to generate ATP
aerobic respiration
the process of oxidizing water to capture chemical energy and generate oxygen
photosynthesis
the conversion of carbon dioxide to organic compounds (particularly glucose)
carbon fixation
a reaction in which electrons are transferred from a compound of lower reduction potential (more negative) to one of higher reduction potential (more positive)
redox reaction
(oxidation-reduction)
_____ refers to a collection of matter in a defined space while _____ refers to everything else
system refers to a collection of matter in a defined space while surroundings refers to everything else
a system in which matter and energy are freely exchanged with the surroundings
open system
a system in which energy is exchanged with the surroundings but matter is not
closed system
a system in which neither matter nor energy are exchanged with the surroundings
isolated system
what is the 1st law of thermodynamics?
1st law: energy cannot be created or destroyed, only converted from one form to another
ΔE = Efinal − Einitial = q − w
a rxn that gives off heat is called _____ and has a (-/+) ΔH value, while a rxn that absorbs heat is called _____ and has a (-/+) ΔH value
a rxn that gives off heat is called exothermic and has a (-) ΔH value, while a rxn that absorbs heat is called endothermic and has a (+) ΔH value
what is the 2nd law of thermodynamics?
2nd law: in the absence of an energy input, all spontaneous processes in the universe tend toward dispersal of energy (disorder = entropy)
ΔSuniverse = ΔSsystem + ΔSsurroundings > 0
a measure of the spontaneity of a rxn
Gibbs free energy (G)
ΔG = ΔH − TΔS
a weak non-covalent bond in which hydrogen is shared b/w 2 electronegative atoms
hydrogen bond
what is the bond strength of a single H-bond?
20 kJ/mol
weak interactions b/w oppositely charged atoms or groups
ionic interactions
weak interactions b/w the dipoles of nearby electrically neutral molecules
van der Waals interactions
a type of weak interaction due to the tendency of hydrophobic molecules to pack close together away from water
hydrophobic effects
a difference in pressure across a semipermeable membrane caused by osmosis across the membrane
osmotic pressure
the diffusion of solvent molecules from a region of lower solute [] to one of higher solute []
osmosis
molecules are attracted to both polar and nonpolar environments
amphiphilic molecules
an aqueous solution that resists changes in pH because of the protonation or deprotonation of an acid-base conjugate pair
buffers
a weak acid with more than one dissociable H+
polyprotic acid
a.a. within a polypeptide chain
residues
short polypeptides that contain less than 40 a.a.
oligopeptides/peptides
each polypeptide chain is called a protein _____
each polypeptide chain is called a protein SUBUNIT
the pH at which a given a.a. has no net charge
isoelectric point (pI)
an electrically neutral but dipolar molecule that contains both positive and negative charges
zwitterion
molecules with the same molecular formula and atomic connectivity, but different 3D orientation of their atoms
stereoisomers
stereoisomers that are mirror images of each other and whose structures cannot be superimposed
enantiomers
enzyme that catalyzes the hydrolysis of a peptide bond
protease
refers to the a.a. sequence, which determines how the polypeptide backbone folds into an energetically stable 3D structure
primary (1°) structure
refers to the regular repetitive arrangement of local regions of the polypeptide backbone
secondary (2°) structure
what are the 3 major secondary structures in proteins?
- α helices
- ß strands
- ß turns
- a graphical representation of the polypeptide backbone associated with a protein’s secondary strcuture
- can use this diagram to depict common folds and patterns in proteins
(cartoon) ribbon diagram
includes the spatial location (complete arrangement) of all the atoms in the polypeptide chain
tertiary (3°) structure
the structure of a protein complex containing more than one polypeptide chain
quaternary (4°) structure
collections of secondary structures that describe the spatial arrangement of a polypeptide chain
protein folds
think of folds as a description of the path the polypeptide follows in 3D space
what are the different ways to view 3D structures of proteins?
- space-filling model
- ball-and-stick model
- backbone model
- cartoon ribbon diagram
- each atom is shown as a solid sphere
- shows the overall shape of the protein and the surface exposed to aqueous solvent
- excellent to show surface ligand binding
space-filling model
an excellent model to show bonding arrangement in proteins
ball-and-stick model
- model that shows the general course of the polypeptide chain
- difficult to see secondary structures
backbone model
- model that concentrates on the backbone of the polypeptide chain
- a.a. side chains are eliminated
- can see into interior of the protein
- can be used to depict common folds and patterns in protein
cartoon ribbon diagram
- a right-handed helical conformation of a polypeptide chain
- one of the most common elements of protein secondary structure
α helix
a 2° structure of proteins consisting of an extended polypeptide chain with side chains positioned above and below it
β strands
a common secondary structure of proteins formed from β strands hydrogen-bonded together (b/w backbone NH and CO groups on separate strands)
β (pleated) sheets
where is the H-bond found in α-helices?
- intrastand H-bond found b/w residues n & n+4
- b/w carbonyl oxygen (n) and H atom attached to nitrogen in peptide bond located 4 a.a. away (n + 4)
hydrogen bonds in α helix: measurements
- length of H-bonds b/w N and O: 2.8 Å
- 3.6 residues per turn
- pitch of 5.4 Å
which a.a. can’t be a side chain in an α-helix structure? why?
PROLINE
nitrogen in ring lacks a hydrogen and does not contribute to H-bonding; rigid ring also restricts confirmations that can be adopted
when is an amphipathic α helix generated?
when a.a. with hydrophilic or hydrophobic properties are positioned every 3-4 residues along the polypeptide backbone
a β sheet structure in which adjacent β strands are oriented in opposite directions with regard to amino to carboxyl termini
antiparallel β sheet
a β sheet structure in which β strands lie in the same amino to carboxyl orientation
parallel β sheets
the hydrogen bond arrangement is more stable for _____ β sheets than for _____ β sheets
the hydrogen bond arrangement is more stable for ANTIPARALLEL β sheets than for PARALLEL β sheets
*why antiparallel β sheets are more common in proteins
a turn or a loop in a polypeptide chain that connects 2 β strands in an antiparallel β sheeet
β turn
what is the difference b/w type I and type II β turns?
type I: carbonyl oxygen b/w 2nd and 3rd residues is oriented inward
type II: carbony oxygen faces outward
- irregular segments that connect elements of secondary structures
- usually range from 6 to 20 residues in length
loops
why are secondary structures common in proteins?
hint: think angles
secondary structures are common because they minimize the steric hindrance of the side chains
which a.a. have the highest propensity to occur in α helices?
AH: Eat Ass Like My Queen/King/Royal Highness Is Watching for Fun
alpha helices
E (glutamate)
A (alanine)
L (leucine)
M (methionine)
Q (glutamine)
K (lysine)
R (arginine)
H (histidine)
I (isoleucine)
W (tryptophan)
F (phenylalanine)
which a.a. have the highest propensity to occur in β strands?
BS: Lil Matt Vader Is Your Cousin? Word! Fun Times
beta strands
L (leucine)
M (methionine)
V (valine)
I (isoleucine)
Y (tyrosine)
C (cysteine)
W (tryptophan)
F (phenylalanine)
T (threonine)
which a.a. have the highest propensity to occur in β turns?
BuT: Each King Got New Pants Sent Down
beta turns
E (glutamate)
K (lysine)
G (glycine)
N (asparagine)
P (proline)
S (serine)
D (aspartate)
what are the 4 general classes of protein structures?
- predominantly α helix
- predominantly β sheets
- α/β combined (Intermixed helices and strands)
- α + β (helical regions adjacent to sheets)
an independent folding module within a polypeptide chain
domain
a small but distinct structural unit of a protein fold
motif
contains 4 α-helices linked together (one of the most common protein folds)
four-helix bundle
consists of 4 or more β strands linked together to form β-sheet structures
Greek key fold/motif
- consists of 2 regions of alternating α helices and β strands that fold together into a compact structural domain
- found in several proteins that bind nucleotides
Rossmann fold
an alternating α helix/β strand fold first identified in the glycolytic enzyme triose phosphate isomerase
TIM barrel fold
- compact domain of about 200 a.a. residues
- contains eight or more radial β strands that form interior of protein (surrounded by eight α helices that face outward)
TIM Barrel fold
a large protein fold (300 a.a.) containing 3 distinct structural components that are found in some proteins that bind to membrane-associated biomolecules
FERM Domain fold
what are the structural components of the FERM domain fold?
FA/FC: mostly β sheets
FB: contains only α helices
what are the 4 proteins where the FERM domain fold is found?
- Band 4.1
- ezrin
- radixin
- moesin
tertiary structures can be stabilized by _____ and _____
tertiary structures can be stabilized by DISULFIDE BONDS and METAL IONS
the number and overall arrangement of the polypeptide chains w/in multi-subunit protein complexes
quaternary structure
a protein complex containing two identical protein subunits encoded by the same gene
homodimer
a protein complex with 2 subunits derived from distinct polypeptides (different gene products)
heterodimer
how do multiple protein subunits (4º) provide increased functional to proteins?
- by providing structural properties not present in individual subunits
- by providing a mechanism for regulation of protein function through conformational changes that alter the protein subunit interface
- significantly increasing the efficiency of biochemical processes by bringin linked functional components into close proximity
a homodimer of 2 helical polypeptides wrapped around each other to form a coiled coil
keratin
(a fibrous protein)
a fibrous protein consisting of 3 subunits (2 heavy chain, 1 light), one of which consists entirely of β sheets
silk fibroin
a protein complex consisting of long helical subunits that are intertwined to form a right-handed triple-helix fiber
collagen
a protein that facilitates the formation of stable 3D structures through the process of protein folding
chaperones
what are the 3 main functions of chaperone proteins?
- help newly synthesized proteins fold properly
- rescue misfolded proteins
- disrupt protein aggregates
a denaturing condition in which 50% of the proteins are fully folded and 50% are fully unfolded (no partially folded/unfolded proteins)
transition curve midpoint (Tm)
an enzyme that cleaves RNA, also the first protein shown to be capable of denaturing and refolding in vitro
ribonuclease A (RNAaseA)
an intermediate stage in a model of globular protein folding in which hydrophobic residues first form the interior of the protein
molten globule
a protein folding model which proposes that hydrophobic residues first form the interior of the protein through loosely defined tertiary structures (molten globules)
hydrophobic collapse model
a protein folding model which proposed that local 2° structures form independently in the 1st phase, leading to the formation of 3° structures in 2nd phase
framework model
a protein folding model which proposes that local 2° structures form before 3° structures, driven by localized interactions b/w 2° structures
nucleation model
the two most common type of chaperone proteins are the _____ and the _____, which bind to misfolded proteins and use ATP hydrolysis as an energy source to facilitate correct folding
the two most common type of chaperone proteins are the CLAMP-TYPE and the CHAMBER-TYPE, which bind to misfolded proteins and use ATP hydrolysis as an energy source to facilitate correct folding
a group of proteins that provide a way for the cell to recover from heat denaturing by helping proteins refold when the temperature returns to normal
heat shock family (clamp-type chaperone proteins)
a well-characterized clamp-type chaperone protein
Hsp70
named from Its molecular mass of 70 kDa
consists of 14 identical polypeptide subunits arranged in 2 rings of 7 subunits each that houses a protein folding chamber
GroEL component of chaperonins (Hsp60)
what are the 3 protein domains of the GroEL component of chaperonins/Hsp60?
substrate binding domain: form walls of protein folding chamber
intermediate domain: form walls of protein folding chamber
ATP binding domain: separates upper and lower chamber; controls opening and closing of chamber
- 7 identical subunits that functions as the cap for the chaperonin/Hsp60 complex
- binds to only one end of the chaperone complex at a time
GroES component of the GroEL-GroES complex
what are the possible causes for protein folding diseases?
- due to mutations in the protein-coding sequence of the gene
- accumulated misfolded proteins that contain no a.a. changes
an enzyme found in bioluminescent organisms that catalyzes a reaction of luciferin to generate visible light (one of the earliest biochemical assays)
luciferase
an isotonic suspension of lysed cells, used for protein isolation and identification
cell extract/cell homogenate
what are the most commonly used homogenization techniques?
sonication: disrupts cell membranes through vibrational effects of ultrasonic waves
shearing: forcing cells through a small opening
incubation of cell sample with mild detergents: disrupts cell membranes
- a process that separates particles of different sizes or densities by spinning solution samples in a rotor at high speeds
- separates large molecules into fractions called pellet and supernatant
centrifugation
the total amount or activity of a target protein, divided by the total amount of protein in the sample
specific activity
what are the four fractions that can (possibly) be obtained from a eukaryotic cell extract after centrifugation?
nuclei
mitochondria
plasma membrane components
cytosol
a protein separation method that involves adding increasing amounts of a saturated ammonium sulfate solution to the protein solution
salting out
a diffusion-based process that uses a semipermeable membrane to allow small molecules to cross the membrane but not large molecules (usually proteins)
dialysis
a technique that separates proteins on the basis of differential physical or chemical interactions with a solid gel matrix
column chromatography
what are the 3 types of column chromatography?
gel filtration
ion-exchange
affinity
a column chromatography method that uses porous hydrocarbon beads to separate proteins on the bases of size
gel filtration chromatography (aka size-exclusion)
a protein purification method that exploits charge differences b/w proteins
ion-exchange chromatography
what are the 2 commonly used ion exchange matrices in ion-exchange chromatography?
(+) charged anion-exchange matrix called DiEthylAminoEthyl (DEAE) cellulose
(-) charged cation-exchange matrix called CarboxyMethylCellulose (CMC)
a buffer solution with a high [] of an appropriate competing ion to dispalce the bound protein (used in ion-exchange chromatography)
elution buffer
a column chromatography method that exploits specific binding properties of the target protein to separate it from other cellular proteins that lack this binding function
affinity chromatography
a biochemical technique that separates proteins on the basis of charge and size
gel electrophoresis
a gel electrophoresis technique that uses a solid-support molecular sieve made of polyacrylamide to separate molecules in an electric field on the basis of charge and size (on the basis of their mass)
polyacrylamid gel electrophoresis (PAGE)
- an amphipathic molecule used in gel electrophoresis to give protein a net negative charge (and migrate toward anode (+ charge) at bottom of buffer tank)
- denatures the protein
sodium dodecyl sulfate (SDS)
_____ proteins migrate faster through the buffer-saturated polyacrylamide gel matrix than _____ proteins
SMALL proteins migrate faster through the buffer-saturated polyacrylamide gel matrix than LARGE proteins
_____ proteins resolve well in gels with low percentage of polyacrylamide, while _____ proteins resolve best in high percentage polyacrylamide gels
LARGE proteins resolve well in gels with low percentage of polyacrylamide, while SMALL proteins resolve best in high percentage polyacrylamide gels
a type of polyacrylamide gel electrophoresis that separates proteins on the basis of charge as a function of pH
isoelectric focusing
for isoelectric focusing, at a pH below its isoelectric point, the protein has a net _____ charge, while at a pH above its isoelectric point, the protein has a net ____ charge
for isoelectric focusing, at a pH below its isoelectric point, the protein has a net POSITIVE charge, while at a pH above its isoelectric point, the protein has a net NEGATIVE charge
a protein separation technique that combines isoelectric focusing and SDS-PAGE to separate proteins on the basis of both pI and molecular mass
2D PAGE
uses the fluorescent dyes Cy3 and Cy5 to distinguish 2 protein samples run on the same 2D PAGE gel
2D differential in-gel electrophoresis (2D DIGE)
a protein sequencing method that modifies the N-terminal acid tagging reaction by using phenylisothiocyanate (PITC)
doesn’t disrupt the rest of the polypeptide chain
Edman degradation
how is Edman degradation better than Sanger’s old method?
(for protein sequencing)
Edman degradation does not require the input of additional protein after each round of cleavage
what is a limitation of Edman degradation?
it is not possible to obtain the sequence of a polypeptide longer than 50 a.a. in a single set of reactions
protease enzymes that cleaves a polypeptide chain on the carboxyl side of lysine or arginine
trypsin
protease enzyme that cleaves a polypeptide chain on the carboxyl side of tyrosine, tryptophan, or phenylalanine
chymotrypsin
trypsin cleaves polypeptide chain on carboxyl side of which a.a.?
lysine and arginine
chymotrypsin is a protease enzyme that cleaves the polypeptide chain on the carboxyl side of which a.a.?
aromatics (tyrosine, tryptophan, and phenylalanine)
protease enzyme that cleaves polypeptide chain on carboxyl side of methionine
cyanogen bromide
cleaves the carboxyl side of aspartate and glutamate
S. aureus
a method of measuring the mass-to-charge ratio of molecules, which is then used to deduce the molecular mass
mass spectrometry
- a method for preparing proteins for mass spec and releases polypeptides that have been cleaved by trypsin
- generates a highly charged molecule in the gas phase
elctrospray ionization (ESI)
a method for generating peptide ions for mass spec in which tryptic fragments are released as charged molecules after exposure to laser flash
matrix-assisted laser desorption/ionization (MALDI)
- a method of structure determinataion that detects nuclear spin properties of certain atoms (1H, 13C, 15N) to deduce their relative locations
- gathers info on relative positions of certain atoms in a protein solution
NMR spectroscopy
provides a snapshot in time of protein structure because it gives atomic positions in a static protein crystal lattice
X-ray crystallography
a procedure that determines the phases of diffracted X-rays by comparing the to X-ray diffraction from a crystal containing an electron-rich element
isomorphous element
