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Cell Bio Exam 1
Terms in this set (71)
what reducing agent is often used in SDS Page?
meraptoethanol - breaks disulfide bonds
what does SDS PAGE stand for?
Sodium dodecyl sulfate polyacrylaminde gel electrophoresis
What does "pro", "eu" and "karyon" stand for?
pro-before, eu-true, karyon-nucleus
bonds in order from strongest to weakest
covalent, noncovalent (ionic bonds, hydrogen bonds, van der waals forces, hydrophobic interactions)
Ionic bonds are synonymous with
Why are electrostatic interactions weak in water?
there charges are shielded
How are hydrogen bonds formed?
a hydrogen atom is sandwiched between two electron-attracting atoms (N or O usually)
Why are hydrogen bonds weak in water?
Molecules that can hydrogen-bond can also h-bond with H2O
What causes van der waals forces?
at short distances, there may be forces due to fluctuating electrical charges
What is the driving force of hydrophobic molecules being held together?
This minimizes their disruptive effects on the H-bonding of water
Structure of an amino acid
carboxyl group, amino group, R-group and hydrogen. both ends are polarized at a pH of 7. NH3+ and COO-
Lysine (Lys) is a basic, hydrophilic amino acid with a hydrocarbon chain and NH3+ on the R-group
Serine (Ser) is an uncharged polar molecule with an -OH on the end of the R-group
How are amino acids linked?
By peptide bonds between the N(+) terminus of one amino acid and the Carboxyl terminus (-) of another. There is no rotation about a peptide bond
4 levels of protein structure
1) primary- amino acid sequence linked by peptide bonds
2) secondary- alpha helices and beta sheets formed by hydrogen bonding
3) tertiary- overall shape formed over long stretches of polypeptide by van der waals forces
4) quaternary- more than one polypeptide chain, linked by disulfide bonds
part of the polypeptide that can fold independently of the rest of the protein into a compact, stable structure
How often is there a hydrogen bond in alpha helices?
Every fourth peptide bond, there is a h-bond between N-H and C=O groups in the the polypeptide backbone
Why are disulfide bonds often not in the cytosol?
Reducing agents break the S-S bonds. (oxidizing agents form them)
Difference between interchain disulfide bond and intrachain disulfide bond?
Interchain- between two different polypeptides
Intrachain- within a single polypeptide
Where is the information needed to fold a protein found?
In the amino acid sequence! This determines the 3-D structure, allowing it to spontaneously refold after being denatured.
How to denature a protein
treat with solvents that disrupt noncovalent interactions, such as urea or heat
Main two roles of proteins
Structure and enzymes (chemical reactions necessary for life would be too slow without proteins)
What is the role of SDS?
SDS is a detergent that binds hydrophobic regions of protein so that polypeptide chains unfold and release from lipids and other proteins. It is a negatively ionic detergent that masks the proteins intrinsic charge and makes them negative, so that when an electric field is applied they will migrate towards the positive electrode
Name of a reducing agent that breaks disulfide linkages
Why don't larger proteins, who have more negative SDS bound to them, migrate faster than smaller proteins?
The gel acts as a molecular sieve, where smaller molecules migrate faster because they are less impeded, are experiencing less drag.
Identifying UNKNOWN proteins from organisms with genomes that are fully sequenced
1) Trypsin (protease) cleaves the peptide
2) MALDI (ion source)- TOF (time of flight) find the mass per charge ratio. Ionized peptides are accelerated by an electric field towards a detector
3) protein database searched for matches to identify the peptide
Immunoblotting or Western Blotting
Finding A SPECIFIC protein in a mixture of proteins
1) proteins are separated by SDS-PAGE
2) transfer proteins from the gel to a nitrocellulose membrane using a strong electric current
3) soak membrane in solution of a labeled antibody to reveal the protein of interest (the marker often emits light)
IF a protein is associated tightly with another protein, the partner will precipitate as well when the target protein immunoprecipitates
What is the limit of resolution and what does it depend on?
Limit of resolution- the limiting separation at which two objects can appear distinct from one another
1) the wavelength of the emitting light
2) the numerical aperture (light gathering, angle of light and refractive index on the medium)
size of a typical animal cell
light microscope resolution
limit of resolution of a light microscope is set by what?
the wavelengths of visible light (400nm violet 700nm red)
Equation for Resolution
0.61 (wavelength)/nsin(theta) n=refractive index of the medium, theta is 1/2 the angular width of the cone rays (max of 1), wavelength is the wavelength of the emitted light rays
What yields the best resolution?
The smaller the R value, the better the resolution! So you want a large numerical aperature and a smaller wavelength!
Phase alterations through the specimen due to denser and lighter parts of the cell are translated into bright changes
Differential Interference (Nomarski)
highlights edges where there is a steep change of refractive index
How are phase contrast and nomarski similar?
They both increase phase differences so waves are more nearly out of phase. Changing the phasing of light waves
specimen is lit from the side and only scattered light is seen
coupling fluorescent dyes to antibodies for highly specific binding. can use different dyes to observe multiple proteins
an unlabeled primary antibody is detected with a labeled secondary antibody
How does the wavelength of fluorescent molecules changed from absorption to emission?
The molecules absorb light and electrons are excited at one wavelength, and then electron returns to ground state and emits of photon of a LONGER wavelength (lower energy)--Fluorescence occurs
Describe the two filters of fluorescence microscopes
1) only passes wavelengths that excite the particular fluorescent dye
2) only passes wavelengths when the dye fluoresces
(there is also a beam-splitting mirror in between these two filters)
why are some images from conventional fluorescent microscopes blurry?
illuminated parts of the specimen that are above and below the plane of focus all contribute to "OUT OF FOCUS" light
Confocal Fluroescence Microscopy
laser focuses at a single point and depth and then scans the specimen. Computer creates an image. It excludes out of focus light to retrieve a clear image
What is the advantage of using fluorescent proteins?
They are protein molecules that are inherently fluorescent. Can monitor gene expression or find a peptide location in a cell component. Introducing this gene does not change the function of the protein, and therefore can be used to study living cells over time.
Fluorescence Resonance Energy Transfer:
can find the interactions between two proteins in a living cell
1) the proteins are attached to different color variants of GFP
ex) if a protein is excited with violet light and emits a blue light which then transfers its energy to the green protein and emits green light, the proteins are interacting. If only blue light was emitted than the proteins aren't interacting.
Why are electron microscopes better than light microscopes?
The wavelength of an electron is very small, which allows for a much smaller resolving power (0.05nm).
Transmission Electron Microscope
TEM- larger and upside down, air is pumped out of the column in a vaccuum so that the electrons don't interact with air. The specimen is stained with electron dense matter which scatters some electrons and the others form an image. Dense regions of the specimen appear dark.
Immungo-gold electron microscopy
Can be used to detect specific proteins
1) attach a colloidal gold particle to the secondary antibody
2) the gold is electron-dense and seen as a black dot
3) use different antibodies and sizes of gold to locate different proteins on the surface of a specimen
Scanning Electron Microscopy (SEM)
3-D structure of surface protein, can form images of the surface, not the subcellular organelles
Co-translational protein folding
Proteins are being folded as they are being synthesized. After N-terminal domain emerges, it only takes a few seconds to form into secondary structures (alpha and Beta). The complete folding of protein occurs after release from the ribosome. Slow process of adjusting side chains before the protein in at it's mature conformation.
Name for initial structure of proteins that are folding
What does exposed hydrophobic residues indicate?
That a protein has been incorrectly folded or there is an off-pathway configuration.
can identify incorrectly folded proteins by ~4-5 amino acids with hydrophobic surfaces exposed. Two major types: Hsp70 and Hsp60. They can bind and hydrolyze ATP.
heat shock proteins
-acts early in life, often before the protein leaves the ribosome
-binds target protein, ATP hydrolysis causes a tighter association between Hsp70 and the protein, then the REBINDING of ATP releases the target protein and allows it to REFOLD
-by non-specifically binding to the hydrophobic regions of the protein, Hsp70 also prevents aggregation (that can be harmful)
-often delivers misfolded proteins to Hsp60
-acts after the protein is fully synthesized
-is a protein complex (multimeric)
-forms an isolated chamber for refolding
-ATP binding and a cap releases the protein into the chamber
-ATP hydrolysis weakens the binding of the cap and additional binding of ATP ejects the cap
Example of when misfolded proteins can be detremental to the organism
Alzheimer's disease is thought to be caused by aggregation of neuronal proteins.
Post-translational modifications of proteins
Modifications by covalent attachment of molecules to R-groups:
How does phosphorylation effect net charge of a protein?
Phosphate carries two negative charges
What amino acids does phosphorylation occur on?
Amino acids with -OH R-groups:
3) Tyrosine (Y)
Energy cost of phosphorylation
1 ATP is used in phosphorylation
the addition of a phosphate by a protein kinase releases a lot of free energy
76 AA long
7 lysines, including K48 and K11
1) C-terminal of ubiquitin in activated by activating enzyme (E1)
2) E1 passes activated ubiquitin to (E2) c-terminal glycine bound to it's active site
3) degradation signal on target protein allows it to bind a specific E3
4) C-terminal of ubiquitin binds the lysine side chain (if K11 or K48 signals for degradation)
5) another ubiquitin may also bind forming a polyubiwuitin chain
bond that connects the glycine and lysine in ubiquitin-signal protein
when is a protein signaled for degradation?
K-11 and K-48 linked poly-ubiquitinated proteins are degraded in the PROTEASOME
Structure of Proteasome
ubiquitin hydrolase to cleave the poly-ubiquitin chain for recycling
unfoldase subunits that unfold the target protein (ATP hydrolysis causes the conformational change that pulles the proteins into the core of the proteasome)
N-end rule of protein stability
N-terminus by cleaving of a single peptide bond destabilizes the protein and calls for degradation
resembles sphingolipid, but has a sugar attached instead of a phosphate-linked head group. Derivative of sphingomyelin
polar head group with phosphate, glycerol and 2 long fatty acid chains
Flip-flop movement of lipids in bilayers
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