-
What is viewed with freeze fracture?
ultrastructure
-
Explain freeze-fracture process and what is seen
- a. small pieces of tissue put on metal disk and frozenà mounted on cool stage in vacuum chamber and the frozen tissue block is struck by knifeà 2 pieces
- i. Deviations in fracture plane with elevations, depressions, ridges that reflect the contours of the protoplasm traversed.
- 1. surface gives info about contents of cell
-
How to make the specimen visible in freeze fracture?
- a. to make visibleà replication process by using the fractured surface as a template on which a heavy-metal layer is deposited to provide shadows that accentuate local topography
- a. A carbon layer is then deposited on top of the metal layer to cement the patches of metal into a solid surfaceà tissue thawed, removed, discarded
- i. only replica used
-
Explain the look of the replica
- a. variations in thickness of metal cause variations in number of penetrating electrons to reach viewing screen
- i. less resistanceà more access to cell membrane
- 1. well suited for examining lipid bilayer
-
a. Freeze-fracture replication by itself is good, but even better with __. Why?
freeze etching
- i. frozen, fractured specimen exposed to a vacuum at elevated temperature for a few minutes, during which a layer of ice sublimes from exposed surface
- 1. once some ice removed, surface of the structure can be coated with heavy metal and carbon to create a metallic replica that reveals both the external and internal structure
-
a. development by __ of deep-etching techniques, in which greater amounts of surface ice are removed, allowed look at cellular organs
John Heuser
-
Membrane Fluidity and isolation?
- i. difficult due to soluble form
- 1. removal requires detergent, which are amphipathic, with a polar end and nonpolar hydrocarbon chain
- a. detergents can sub for phospholipids in stabilizing integral proteins while rendering them soluble in aqueous solution
-
i. Once proteins have been solubilized by detergent, various analyses can be carried out to determine the protein’s __(3)__, etc.
amino acid composition, molecular mass, amino acid sequence
-
i. hard to isolate crystals for __, but one of the first membrane proteins whose entire 3D structure was determined by __
1. this protein—__—has __subunits with __
- X-ray crystallography
- X-ray crystallography
- bacterial photosynthetic reaction center
- three
- 11 membrane-spanning alpha helices
-
i. most membrane proteins have not been as amenable to crystallization as the photosynthetic reaction center. Why not?
- 1. among the obstacles, many membrane proteins
- a. are present in low numbers per cell
- b. are unstable in detergent-containing solutions required for their extraction
- c. prone to aggregation
- d. heavily modified by glycosylation and can’t be expressed as recombinant proteins in other types of cells
-
i. some technical difficulties in preparing __ overcome by new methodologies and laborious efforts
ii. in some cases, __ of a membrane protein are found to be better suited to forming the ordered arrays of molecules that make up a __
- membrane protein crystals
- mutant versions
- crystal lattice
-
i. in other ways, crystallization has been achieved by __linking the membrane protein to other molecules, often small __
1. these added elements __ the structure of the target protein and increase the __that can participate in __contacts required for lattice formation
- covalently
- soluble proteins
- stabilize
- number of sites on the surface of the protein
- intermolecular
-
Identifying Transmembrane Proteins
1. computational analysis of its __, which is readily deduced from the __ of an isolated gene
a. the segments within the membrane (transmembrane) have simple structure
i. string of about __ that span the core of the lipid bilayer as an __
- amino acid sequence
- nucleotide sequence
- 20 nonpolar amino acids
- alpha helix
-
1. chemical structure of a single transmembrane helix is 2D structure of __,the major integral protein of the erythrocyte plasma membrane
a. of the 20 amino acids that make up the lone __ of a __monomer, all but three have __
i. exceptions: serine and threonine (non-charged, polar, residues)
- glycophorin A,
- alpha helix
- glycophorin
- hydrophobic side chains
-
1. __ may also appear in transmembrane helices, but they tend to be near __and to be accommodated in ways that allow them to fit into their __
- fully charged residues
- one of the ends of the helix
- hydrophobic environment
-
1. knowing the amino acid sequence of an integral membrane protein, we can usually identify the __segments using a __, in which each site along a polypeptide is assigned a value that provides a measure of the __of the amino acid at that site as well as that of its neighbors
- transmembrane
- hydropathy plot
- hydrophobicity
-
a. this provides a “__” of the __of short sections of the polypeptide, and guarantees that one or a few polar amino acids in a sequence do not alter the profile of the entire stretch
- running average
- hydrophobicity
-
i. hydrophobicity of amino acids can be determined using __
- 1. lipid solubility
- 2. energy required to transfer them from a nonpolar medium into an aqueous medium
-
1. transmembrane segments are usually identified as a __that extends well into the hydrophobic side of the spectrum
a. a reliable prediction concerning the orientation of the transmembrane segment within the bilayer can be made by examining the __
i. those parts of the polypeptide at the __flank of a transmembrane segment tend to be more __charged than those at the __flank
- jagged peak
- flanking amino acid residues
- cytoplasmic
- positively
- extracellular
-
1. not all integral membrane proteins contain transmembrane __
a. a number of membrane proteins contain a relatively large channel positioned within a circle of __ organized into a barrel and have only been found in the outer membranes of __, __, and __
- alpha helices
- membrane-spanning beta strands
- bacteria, mitochondria, and chloroplasts
-
a. Experimental Approaches to Identifying Conformational Changes within an Integral Membrane Protein
i. to determine which sites within transmembrane domains are accessible to aqueous environment and how this changes as the protein carries out is function, __ can be used by using specific changes into the gene that encodes the protein
site-directed mutagenesis
-
a. determining __ between specific __ in a membrane protein is another approach to learning about dynamic events that occur as a protein carries out its function
i. one way to learn distance between selected residues in a protein is to introduce __ whose properties are sensitive to the distance that separate them
- spatial relationships
- amino acids
- chemical groups
-
1. __are chemical groups that contain an unpaired electron, which produces a characteristic spectrum when monitored by a technique called __
- nitroxides
- electron paramagnetic resonance spectroscopy
-
a. a __group can be introduced at any site in a protein by first __that site to a __and then attaching the __to the__ group of the __.
i. this was used to discover the conformational changes that occur in a membrane protein as its channel is opened in response to changes in the __of the medium
- nitroxide
- mutating
- cysteine
- nitroxide
- –SH group of the cysteine residue
- pH
-
a. a technique called __can also be used to measure changes in the distance between labeled groups within a protein
FRET
-
a. Peripheral Proteins
i. associated with membrane by __
can be solubilized by __ with __ that weaken the __ holding peripheral proteins to a membrane
- weak electrostatic bonds
- extraction
- high-concentration salt solutions
- electrostatic bonds
-
i. best studied peripheral proteins are located on the __surface of the plasma membrane, where they form a __ that acts as a __
these proteins provide __ for the membrane and function as an __for __
- internal (cytosolic)
- fibrillar network
- membrane “skeleton”
- mechanical support
- anchor
- integral membrane proteins
-
i. other peripheral proteins on the internal plasma membrane surface function as __, __, or __that transmit transmembrane signals
ii. have dynamic relationship with the membrane, being __to the membrane or __from membrane
- enzymes
- specialized coats
- factors
- recruited
- replaced
-
a. Lipid-Anchored Proteins
i. numerous proteins on the external membrane are bound by small, complex __linked to a molecule __embedded in the __ of the lipid bilayer
1. peripheral membrane proteins containing this type of __are __
- oligosaccharides
- PI
- outer leaflet
- glycosyl-phosphatidylinositol linkage
- GPI-anchored proteins
-
i. On the cytoplasmic side, proteins are anchored to the membrane by one or more long __ embedded in the __of the lipid bilayer
- hydrocarbon chains
- inner leaflet
|
|