Week 1: 1.3-1.5: Cells, Membranes, Membrane Proteins Flashcards
What are the components of the Cell Theory?
- Cells are the basic unit of life
- all living things are made of cells
- cells arise only from other cells
What are the three main forms of life on earth?
- Eubacteria
- Archaebacteria
- Eukaryotes
What two types of prokaryotes are there?
- Eubacteria (e.coli and bacillus)
- archaebacteria
What distinguishes a prokaryotic cell from a eukaryotic cell?
Internal organization: organelles and cytoskeleton.
- Eu: more complex, has a true nucleus, and is compartmentalized. Has membrane bound organelles and has a cytoskeleton
- Prokaryotic: no nucleus, 2 compartments sometime, simple in structure, lack of internal membrane bound organelles, lack of major internal structural systems aka cytoskeleton
- prokaryotes lack membrane bound organelles
What are some of the ways that prokaryotes and eukaryotes are the same?
Structural organization
-surrounded by lipid membrane
-use of nucleic acid to transfer DNA/heritable info
Similar biochemical processes
-metabolic process: all living cells generate ATP to drive metabolism
-Genetic process: all living things use ribosomes and transfer RNA molecule during the conversion of DAN code to protein
-Signaling: use small ions like calcium for communication purposes
What are some of the ways prokaryotes are classified?
Morphology -shape: rods, spheres, spiral, flagellum Diagnostic Stains -gram (+)/(-) Comparison of the nucleotide sequence of the ribosomal rRNA gene
What is a gram-negative bacterium?
Bacteria is surrounded by lipid membrane
What are protozoa? Are they prokaryotic or Eukaryotic?
Eukaryotic simple cell or unicellular organisms that show great complexity.
-protozoa, algae, fungi (yeast)
What is the word cell derived from?
The word “cell” was
first used by Robert Hooke in the 1600’s.
- structures he saw reminded him of small rooms in monasteries where monks lived. These
small rooms were called cells after the Latin word “cellula”, or small room, and so that is why
we call cells “cells”
What are commonalities of all living cells?
- structural features
- biochemical process
- A cell is surrounded by plasma membrane
- A cell has genetic information
What is gram staining?
used to identify bacteria. uses purple or red stains that bind to proteins and peptides creating a stain.
If bacterium is surrounded by an outer membrane will it test gram negative or gram positive?
will test gram-negative b/c lipid membrane does not bind to the stain. Stain only binds to peptide bonds and proteins
If bacteria are enclosed in a coat of peptides & polymeric sugars, aka petidoglycan layer, will it test gram negative or positive?
will test gram-positive
why do some prokaryotes have a second compartment? What is this second compartment called?
- 2nd compartment is called periplasmic space
- sometimes the cytoplasm is surrounded by one double layered lipid membrane aka plasma membrane and sometimes they have two membranes aka the second comparment aka periplasmic space between them.
What are the two compartments that prokaryotes can have?
- cytoplasm or cystolic: contains water-soluble proteins and DNA
- periplasmic space: when a prokaryote has two outer membranes this is the space between them
what is a plasma membrane?
one double-layered lipid membrane that surrounds the cytoplasm
what is meant by a fluid mosaic biomembrane?
- the fluid part are the lipids (which move sideways freely but not across membrane)
- mosaic is the protein floating around (can be bound by other proteins or lipids)
What components make a biomembrane?
lipids, proteins, and carbohydrates
Why are lipids known as amphipathic molecules?
They have a polar head (water-soluble/hydrophilic) and 2 non-polar tails (excluded from water/hydrophobic) = dual solubility
What is the effect of adding cis bonds to a lipid?
One tail typically has one or more cis-double bonds (that is, it is unsaturated), while the other tail does not (that is, it is saturated). Each cis-double bond creates a kink in the tail. Differences in the length and saturation of the fatty acid tails influence how phospholipid molecules pack against one another, thereby affecting the fluidity of the membrane,
Alberts, Bruce. Molecular Biology of the Cell (p. 566). W. W. Norton & Company. Kindle Edition.
What are the three main classes of lipids in biological membranes?
- Phosphoglycerides (phospholipids)
- Sphingolipids
- Sterols
What are some of the effects of adding cholesterol to a biological membrane?
- cholesterol can change membrane fluidity and permeability
- cholesterols saturates membrane meaning the membrane is LESS permeable and LESS fluid
- cholesterol fills the spaces created by unsaturated lipids
- stops gel formation
- shifts the phase transition temp lower.
How can the fluidity of a membrane be altered?
-Temperature
(low temp: gel-like…butter)
(high temp: fluid-like…melted butter)
- adding cholesterol
- Phase transition temperature (Tm)
What is the phase transition temperature?
temp at which half of the membrane is in the gel
phase or half of the membrane is in the fluid phase
What is the molecular structure of a phosphoglyceride?
- Polar (hydrophilic head) made up of choline, phosphate, and 3Carbon glycerol.
- Glycerol is linked (by ester bond) to one or two fatty acid tails
- fatty acid tails are non-polar & hydrophobic
What is the general structure of a sterol?
- OH group making the polar head (hydrophilic)
- Four-ring hydrocarbon (rigid and planar)
- hydrocarbon chain forms the hydrophobic tail
What is the structure of sphingolipids?
Just like phosphoglycerides, the polar, water loving head is made up of choline, phosphate, and in place of glycerol is sphingosine. sphingosine is then attached to a fatty chain and a fatty acid tail (non polar)
List the various motions of lipid molecules
4 types:
1) lateral diffusion (side to side)
2) Rotation (360)
3) flexion/flexing (180)
4) flip-flops (rarely occur b/c they usually need an enzyme like flippase or scamblase to happen)
Why is the fluid mosaic nature of cell membranes important?
Has lipids and proteins. Proteins are the primary way the cell differentiates and specializes a membrane.
The fluid nature of lipid bilayers allows proteins to move from one place in the membrane to another and allows cells to reorganize the membrane as needed
the fluid nature of the membrane implies that cells must have mechanisms for localizing or restricting the movement of the proteins in order to create specialized membrane regions
what structures can you find present in membranes?
lipids and protein complexes
what does it mean when a molecule is saturated vs. unsaturated?
Saturated: single bond, many linked hydrogens, lipids have to be tightly compressed
unsaturated: cis bond, less linked hydrogens, mores space for lipids to arrange themselves
why is the head of a lipid molecule water soluble?
head of lipid is hydrophilic, polar, and has negative/positive charge and so does water meaning it is soluble.
what are glycolipids?
modifications of sphingosine that are common in the cell nervous system and present in exoplasmic face of lipid membranes
what are the predominant lipids in animal membranes?
phosphoglycerides
Which phosphoglyceride has a net negative charge?
phosphatidylserine
Why are flip flops rare?
Flipping involved moving a polar head thorugh the nonpolar membrane. This usually requires an enzyme like flippase and scramblase
what factors can affect fluidity of a membrane?
Temperature
Sterols
Double bonds in hydrocarbons
length of hydrocarbon bonds
What makes membrane less fluid?
Saturated hydrocarbon chains
Longer hydrocarbon tail
Thicker
What makes membrane more fluid?
Unsaturated hydrocarbon chains
shorter hydrocarbon chains
thinnner
What are the different classes of membrane associated proteins?
Intergral proteins: transmembrane proteins
Anchored proteins: cytosolic or non cytosolic
Peripheral proteins: cytosolic and non-cytosolic
What are some of the main differences between alpha helical and beta barrel membrane proteins?
Alpha helical are usually 20-25 amino acids, can shit positions fairly easily, are flexible in forming functional domains, hydrophobicity can be determined
beta barrels are 10 or fewer amino acids in length, are relatively stiff and unchangeable in form, cannot be identified by hydrophobicity (non polar amino acids face out and polar amino acids face in)
What are some of the characteristics of an alpha helical transmembrane domain?
- can go entirely through the membrane before turning around
- can also turn around before going through one leaflet (aquaporin)
- transmembrane helices often interact w/one another
- connected by flexible non-helical linkages
What are the three types of linkages that might be involved in anchoring a protein to a membrane?
Amide linkage
Thioester linkage
Exoplasmic GPI link to protein
What is an aminde linkage?
anchorage
formed between n-terminal glycine and a fatty acid (myristic acid)
What is a thioester linkage?
Forms between a terminal cysteine and fatty acids (palmitoyl or prenyl group)
GPI (glycosylphophatidylinositol)
exoplasmic link GPI attaches to a protein
How could you experimentally distinguish between integral and peripheral membrane proteins?
for anchored and integral you can use a detergent. The detergent isolates protein from membranes and disrupts the membrane
Peripheral can be identified by their weak ionic bond association with membranes. Experiment: use aqueous solutions of varying ionic strength such as high salt concentration.
Using aqueous solutions of varying ionic strengths could help you identify what proteing?
peripheral proteins
Which side of a membrane do you find glycosyalated proteins ?
exoplasmic
the additional of carbonhydrate molecules to proteins happens within the lumen of endoplasic reticulum and golgi apparatus - which usually end up on the outside of the membrane after secretion
What is a glycocalyx, and what are some of its functions?
fuzzy surface taht coats the outside of cell & is made of a layer of carbohydrates
Functions: Cellular processes cell protection cell attachment to outside environment cell to cell recognition
How might you study movement of proteins within a membrane?
FRAP and FLIP
Fluorescent recover after photobleaching
Fluorescent loss in photobleaching
What is the difference between FRAP and FLIP?
FRAP:
- labels proteins with fluorescent probes
- small bleached area
- large unbleached proteins
- immediately - bleached area would have no proteins that were fluorescent
- over time bleached area would move out of bleached zone and unbleached proteins would move into bleached area
FLIP:
- one area of cell bleached repeatedly
- intensity of fluorescence in a neighboring area is monitored instead of fluorescence in the area that’s being bleached
- gradual loss of fluorescence in the area that is not being bleached b/c proteins are moving b/c proteins are moving into that bleached area and being bleached
If you conduct a FRAP experiment using fluorescent labeled membrane protein and observe only 75% recover of the pre-bleach intensity, what does this mean?
- Membrane is not continuous and is divided into separate micro domains
- membrane components can move within the domains but crossing from one domain to another is not frequent
What mechanisms are involved in regulating the position of membrane associated proteins in a polarized epithelial cell?
Cells create a tight junction when they come in contact. Proteins that are positioned on the side of the cell or apical side of the cell are unable to move between tight junctions. Proteins in the apical side can move in their domain but cannot move into the basolateral part of the cell, since that is its own domain. Likewise, proteins in the basolateral part of the cell will not move into the apical side.
How are membrane associated proteins distinguished?
by how they attach to the membrane
How are integral proteins embedded with in a bilayer membrane and attache protein to bilayer?
By hydrophobic interactions
Do anchored and peripheral proteins need to have hydrophobic domains?
No because they don’t interact directly with the membrane
How do integral proteins associate with the membrane?
- Embedded w/in the protein by hydrophobic interactions
- can be associated with one face of the bilayer or cross both lipid layers
How do anchored proteins associate with the membrane?
attached to the membrane through a linkage of some sort. Such as covalent attachments to glycolipids
How do peripheral proteins associate with the membrane?
loosely attached.
-can attached to lipid membrane or another more tightly associated protein
Transmembrane proteins can be categorized as what type of protein?
Integral proteins
- single pass transmembrane
- mulit pass transmembrane
- beta barrel proteins on the outer layer
Why are transmembrane proteins important ?
They allow the inside and outside of the cell to communicate and interact with each other.
Channel forming proteins can be categorized as?
Beta Barrel proteins; and they allow regulated movement of material through a pore that’s in the middle of the barrel called porins
If a protein needs to change it’s size significantly to perform a function will it be an alpha or beta?
Alpha. since beta barrels are rigid it doesn’t allow for protein movement, meaning it won’t allow a protein to move to perform it’s function.
Is it easy to distinguish the hydrophobicity of an alpha helix protein? what about beta?
It is easy to distinguish the hydrophobicity of an alpha helix protein because the helical part that passes through the membrane is mostly non polar amino acids. using a computer program to calculate statistical analysis of the amino acid sequence of proteins and predict the likelihood that a region of proteins is hydrophobic or hydrophilic. It is not easy to distinguish with beta barrels b/c they have nonpolar aminos that face out and polar amino acids that face in - not clear how many exist in a genome.
Are proteins that enter the cytoplasm or external environments on either side of the membrane generally hydrophobic or hydrophilic?
Hydrophobic and normal contain a higher concentration of polar amino acids. Most transmembrane proteins are amphipathic and contain both hydrophobic and hydrophilic regions
transmembrane proteins that need to change their shape in a large way in order to perform a
function, are typically going to made up of what?
multiple alpha helical coils connected to each other.
WHY?
some transmembrane proteins have regions
in their interior which are protected from the hydrophobic membrane by the layers of
surrounding protein. This environment allows protein loops that can be hydrophilic to form
inside the membrane itself and it allows the proteins to make turns within the bilayer. This
greatly increases the flexibility these proteins have in generating functional domains. Because
individual alpha helices are connected by flexible non-helical linkages, the transmembrane
helices can also shift their position relative to each other fairly easily
The simplest arrangement of a transmembrane protein is…?
single alpha helical coil
a single alpha-helical coil of hydrophobic amino acids,
which traverses the lipid bilayer. These proteins can also be covalently linked to lipids within the
membrane, this tends to further stabilize the membrane
protein within the membrane.
Do anchored proteins cross the membrane? How does this affect their activity?
No they do not. This cause their activity to be restricted to one side of the membrane or other.
A protein that associates with membranes through attachment to other proteins is categorized as what kind of protein?
Peripheral protein. Interact with integral proteins by ionic interaction. Can be cytosolic or extracellular
How does one distinguish between peripheral proteins and integral and anchored proteins.
Experimentally you can create aqueous solutions of varying ionic strength, such as high salt concentration. B/c peripheral proteins have weak ionic bonds this would break the bonds. Integral and anchored need detergent to break the bond between them and the membrane since detergent disrupts integrity of membrane itself.
What type of modifications can proteins undergo on the exoplasmic side of the membrane?
addition of further linkages to carbohydrate molecules, like oligo and
polysaccharides, as well as the formation of disulfide bonds, which may stabilize the shape of the
protein. These modifications are generally found only on the exoplasmic proteins and not on
cytosolic proteins and this is because the addition of carbohydrate molecules to proteins happens
within the lumen of the endoplasmic reticulum and the golgi apparatus
Why are disulfide bonds rare within the cytoplasm?
because the internal cytoplasm is a reducing
environment, which breaks disulfide bonds.
Does glycosylation happens to exoplasmic or cytoplasmic proteins?
Exoplasmic: The glycosylation of exoplasmic proteins, produces
a layer of carbohydrates on the outside of the cell, that coats the outer surface of the cell and this
is known as a glycocalyx
Does glycosylation happens to exoplasmic or cytoplasmic proteins?
Exoplasmic: The glycosylation of exoplasmic proteins, produces
a layer of carbohydrates on the outside of the cell, that coats the outer surface of the cell and this
is known as a glycocalyx
How do we study protein lateral diffusion?
Can use cell fusion method.
Two
different types of cells, each with unique membrane associative proteins are fused to create a
single, larger cell. Initially, proteins unique to the individual cells are found in localized areas of
the fused cell, but they rapidly disperse over the entire cell surface
What type of protein movement is common? Which one is rare?
Rotational and lateral movement of proteins is common and movement across the membrane is rare
What are the advantages of using FLIP over FRAP?
the fluorescent proteins which are being looked at or measured are not
the ones that are being exposed to the potentially damaging laser light.
What are some strategies cell use to restrict movement of proteins?
A: aggregation
B: tethered from outside;
C: tethered from inside,
D: tethered by another cell
