BIOCHEM 4 & 5 Flashcards
Life cycle of protein
Translation, Post-translational modifications, Age via oxidation, Degrade into amino acids
Importance of Protein Purification
Detailed and accurate study of protein properties
Living organisms contain thousands of proteins in varying amounts,
so _____________ can interfere with accurate analysis
contaminants
Differences in solubility based on pH, polarity, or salt concentration
Selective precipitation
Stationary phase (beads in columns) separates proteins by size, charge, hydrophobicity, or affinity for ligands
Column chromatography
Uses small, strong silica beads for high resolution separation under high pressure
High-Pressure Liquid
Chromatography (HPLC)
Traditional column chromatography uses _______________ that ____________________
the separation process and_______________.
large beads, slow down, lower resolution
HPLC uses ______________ beads that allow better separation of
proteins due to the ______________________.
tiny & porous silica, higher surface area
In HPLC the small size of the beads means ________________________ are needed to push the liquid through the column, but this pressure results in ____________
and more ____________ protein separation.
higher pressures, faster, precise
Separation by size; larger proteins
move faster
Size-Exclusion Chromatography
In Size-Exclusion Chromatography __________ proteins cannot enter the porous beads used in the stationary phase, so they pass through the column faster than ____________ proteins, which are
delayed as they enter the pores
Larger, smaller
Separation by charge; positive proteins bind to negative beads
Ion-Exchange Chromatography
Separation by hydrophobicity; useful for membrane
proteins
Hydrophobic Interaction Chromatography
Separation of proteins by size; polypeptides
migrate based on molecular mass.
SDS-PAGE
SDS-PAGE is visualized with dyes like _______________________
Coomassie Blue
Separates proteins based on isoelectric point
(pI); used in 2D electrophoresis combined
with SDS-PAGE
Isoelectric Focusing (IEF)
SDS (sodium dodecyl sulfate) binds to proteins in a ratio that is
__________________ to the protein’s size, essentially giving each protein a uniform _______________ ratio.
proportional, charge-to-mass
The separation of proteins in SDS-PAGE depends purely on their _____, not their intrinsic _______________
size, charge or shape
First method for polypeptide
sequencing, used insulin as the model
Sanger Sequencing
Sequential removal of
amino acids from the N-
terminus
Edman Degradation
Edman Degradation is ore efficient but limited
to _________ sequences (_______ residues)
shorter, 5- 30
Edman degradation method has limitations such as its inefficiency with______________ and ___________________
longer peptides, low throughput
__________________________ is faster, more sensitive, and capable of detecting minute posttranslational modifications.
Mass spectrometry
One of the key advantages of mass spectrometry is its ability to detect modifications like _________________, ______________, and ____________________, which
are important for understanding protein function and regulation.
phosphorylation, methylation, glycosylation
Revolutionized protein sequencing, identifying proteins by
their mass and modifications.
Mass Spectrometry
Techniques to vaporize proteins for mass analysis
Electrospray Ionization and MALDI
Allows for analysis of complex peptide mixtures without prior purification
Tandem MS (MS/MS)
In Tandem MS Proteins are first __________ and _______________
ionized, fragmented
__________________ are more susceptible to breaking than ___________________, meaning the primary fragmentation products are peptides of
varying lengths
Peptide bonds, carbon-carbon
bonds
_______________ are weaker than most other bonds in a protein, making them the ________________________ during mass spectrometry.
This selective fragmentation allows for accurate sequencing.
Peptide bonds, primary targets for fragmentation
Goal is to map the entire proteome under diverse
conditions, identifying proteins and their modifications
Proteomics
Facilitates the understanding of protein functions by
comparing sequences and using gene arrays
Bioinformatics
crucial for function, whether in catalysis, motion,
or providing structural integrity
PROTEINS
involves spatial arrangement
(reversible)
Conformation
requires breaking covalent bonds
Configuration
Linear sequence of amino acids
Primary Structure
Local folding into α-helices and β-sheets
Secondary Structure
3D folding of the entire polypeptide
chain
Tertiary Structure
Assembly of multiple polypeptide
chains
Quaternary Structure
Twisted backbone stabilized
by hydrogen bonds, often
amphipathic
Alpha Helix
Proteins are composed of __________________, and their
stereochemistry favors the
formation of ______________________
L- amino acids, right-handed
alpha helices
_________________ are unstable and
rarely form due to steric hindrance and _______________ interactions between amino acids
Left-handed helices, less favorable
Alpha helices are stabilized by
___________________ between the
__________________ and the ______________________ spaced _____ residues apart, giving the helix its regular, coiled structure
hydrogen bonds, carbonyl oxygen, amide hydrogen of amino acids, four
_______________ lacks the hydrogen atom required to form hydrogen bonds in an alpha helix, which makes it a
“_______________________.”
Proline, helix breaker
Its rigid structure introduces a kink or bend in the helix, disrupting the regular hydrogen bonding pattern
Proline
due to its small size
and flexibility, can also
disrupt the helix structure,
although it does not break
the helix as dramatically as
proline
Glycine
Zigzag backbone
stabilized by interstrand
hydrogen bonds
Beta Sheet
Shows permissible
φ(phi) and ψ (psi)
angles for secondary
structures
Ramachandran Plot
Complex 3D arrangement of helices, sheets, and loops
forming functional domains
Tertiary structure
Modular units of structure within a protein, often having
specific functions
Domains
are distinct structural units within a polypeptide, and
each domain can perform a specific function
Protein domains
Domains are ______________, meaning they can fold independently and carry
out their function regardless of the rest of the protein’s structure
modular
This modularity allows proteins to perform complex tasks efficiently
Independent Folding
Proteins with multiple subunits,
such as homodimers or
heterodimers
Oligomeric Proteins
Most energetically favorable and functional state
Native Conformation
Folding Process is a ______________ with the aid of chaperones
Modular process
Proteins fold via an ordered process involving ___________,
_________, and _______________ structures
secondary, tertiary, quaternary
Stabilization Forces
Hydrogen bonds, van der Waals forces, Hydrophobic interactions
are often found in the hydrophobic cores of globular
proteins because their extended structure can pack tightly and
exclude water, stabilizing the overall fold of the protein
Beta sheets
Loops and turns, which are more flexible and irregular, are
typically found on the _________________, where they interact with
the aqueous environment or other molecules
protein surface
Proteins like _______________ and __________________ assist
in proper folding and prevent aggregation
chaperones, disulfide isomerase
High-resolution 3D structure determination
X-ray Crystallography
Used to study proteins in solution
Nuclear Magnetic Resonance (NMR)
Emerging technique for studying large protein complexes
Cryo-Electron Microscopy (Cryo-EM)
