Protein Analysis Microbiology
Escherichia coli: a Gram-negative bacterium of the intestine microbiome, Half 1
FIGURE three.1 ■ Escherichia coli: a Gram-negative bacterium of the intestine microbiome. The envelope contains the outer membrane; the cell wall and periplasm; and the inside (cell) membrane. Embedded within the membranes is the motor of a flagellum. The cytoplasm contains enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, that are folded by chaperones. The nucleoid accommodates the chromosomal DNA wrapped round binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
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Bacterial Cell Construction: What’s seen in Gram-negative & Gram-positive micro organism
Micro organism could be positioned in 2 teams based mostly on the thickness and placement of the cell wall
Gram-negative
Gram-positive
Plasma membrane
Absence of a nucleus
DNA is situated within the nucleiod area
No histone proteins, however DNA-binding proteins current to maintain genomic DNA compact
Plasmids: DNA that’s impartial of the genome.
Flagellum
Biochemical composition of micro organism
Water
Important Ions
Wanted for enzymatic reactions
Small natural molecules: lipids and sugars
Lipids are virtually as considerable as RNA molecules
Discovered within the cell wall
peptidoglycan
Macromolecules: nucleic acids, proteins, fat, & sugars
Aim: Isolate proteins
Objective of cell fractionation is to isolate parts of alternative from a bacterial cell
Step one is cell lysis
EDTA
Sucrose
Lyzozymes
Ultracentrifugation
FIGURE three.2 ■ Fractionation of Gram-negative cells.
Cell periplasm fills with sucrose, and lysozyme breaks down the cell wall. Dilution in water causes osmotic shock to the outer membrane, and periplasmic proteins leak out. Subsequent centrifugation steps separate the proteins of the periplasm, cytoplasm, and inside and outer membranes. Picture
Supply: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
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Aim 2:Protein Analysis
FIGURE three.three ■ Protein Assessment.
A. Gel electrophoresis of complete cell proteins in comparison with outer membrane proteins from cell fractionation. B. Outer membrane proteins are recognized by tryptic digest and mass spectrum Assessment. The ensuing peptide sequence is in contrast with these predicted from genome information.
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FIGURE three.3a ■ Protein Assessment.
A. Gel electrophoresis of complete cell proteins in comparison with outer membrane proteins from cell fractionation.
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FIGURE three.3b ■ Protein Assessment.
B. Outer membrane proteins are recognized by tryptic digest and mass spectrum Assessment. The ensuing peptide sequence is in contrast with these predicted from genome information.
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Understanding the function of a protein
FIGURE three.four ■ Genetic Assessment of FtsZ.
A. E. coli with aspartate (D) at place 45 changed by alanine (A) (D45A) elongate abnormally, forming blebs from the facet, with no Z-rings. Cells with aspartate changed by alanine at place 212 (D212A) elongate to type prolonged nondividing cells that comprise spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Mannequin of FtsZ protein monomer based mostly on X-ray crystallography exhibits the place of the mutant residues, D212A and D45A.
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FIGURE three.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer accommodates stiffening brokers resembling hopanoids. Half the membrane quantity consists of proteins.
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LeuT sodium/leucine cotransporter
Homology to human neurotransmitter sodium sympoters
Has been used as a blueprint to know construction and performance, and pharmacology of NSS human transporters.
FIGURE three.7 (half 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex micro organism.
The protein advanced carries leucine throughout the cell membrane into the cytoplasm, coupled to sodium ion inflow. (PDB code: 3F3E)
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Transport throughout bacterial membranes
Passive diffusion
Membrane proteins
Aquaporins
Permease (lac operon)
Osmosis
Better osmotic stress can result in bacterial cell lysis (seen with sure antibiotics)
Membrane-permeant weak acids and bases: can cross the plasma membrane
Transmembrane ion gradients
FIGURE three.eight ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
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FIGURE three.8a ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
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FIGURE three.8b ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
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NAM and NAG are linked collectively by a β-(1,four)-glycosidic bond
Lysozymes goal this bond
The peptidoglycan monomer may have 5 peptides
As soon as this monomer turns into integrated into the prevailing polymer, four peptides are seen.
FIGURE three.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an connected peptide of 4 to 6 amino acids.
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FIGURE three.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with a number of layers of peptidoglycan, threaded by teichoic acids. A inset: Gram-positive envelope of Bacillus subtilis (TEM). B. Proteobacteria (Gram-negative) cells have a single layer of peptidoglycan coated by an outer membrane; the cell membrane is named the inside membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
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Gram +
FIGURE three.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted within the inward-facing leaflet of the outer membrane. The C-terminal lysine varieties a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
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FIGURE three.20 ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to 6 fatty acids. B. Repeating polysaccharide models of O antigen lengthen from lipid A.
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FIGURE three.20a ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to 6 fatty acids.
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FIGURE three.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide models of O antigen lengthen from lipid A.
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FIGURE three.1 ■ Escherichia coli: a Gram-negative bacterium of the intestine microbiome. The envelope contains the outer membrane; the cell wall and periplasm; and the inside (cell) membrane. Embedded within the membranes is the motor of a flagellum. The cytoplasm contains enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, that are folded by chaperones. The nucleoid accommodates the chromosomal DNA wrapped round binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
*
FIGURE three.2 ■ Fractionation of Gram-negative cells.
Cell periplasm fills with sucrose, and lysozyme breaks down the cell wall. Dilution in water causes osmotic shock to the outer membrane, and periplasmic proteins leak out. Subsequent centrifugation steps separate the proteins of the periplasm, cytoplasm, and inside and outer membranes. Picture
Supply: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
*
FIGURE three.three ■ Protein Assessment.
A. Gel electrophoresis of complete cell proteins in comparison with outer membrane proteins from cell fractionation. B. Outer membrane proteins are recognized by tryptic digest and mass spectrum Assessment. The ensuing peptide sequence is in contrast with these predicted from genome information.
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FIGURE three.3a ■ Protein Assessment.
A. Gel electrophoresis of complete cell proteins in comparison with outer membrane proteins from cell fractionation.
*
FIGURE three.3b ■ Protein Assessment.
B. Outer membrane proteins are recognized by tryptic digest and mass spectrum Assessment. The ensuing peptide sequence is in contrast with these predicted from genome information.
*
FIGURE three.four ■ Genetic Assessment of FtsZ.
A. E. coli with aspartate (D) at place 45 changed by alanine (A) (D45A) elongate abnormally, forming blebs from the facet, with no Z-rings. Cells with aspartate changed by alanine at place 212 (D212A) elongate to type prolonged nondividing cells that comprise spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Mannequin of FtsZ protein monomer based mostly on X-ray crystallography exhibits the place of the mutant residues, D212A and D45A.
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FIGURE three.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer accommodates stiffening brokers resembling hopanoids. Half the membrane quantity consists of proteins.
*
FIGURE three.7 (half 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex micro organism.
The protein advanced carries leucine throughout the cell membrane into the cytoplasm, coupled to sodium ion inflow. (PDB code: 3F3E)
*
FIGURE three.eight ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE three.8a ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE three.8b ■ Frequent medication are membrane-permeant weak acids and bases.
In its charged type (A– or BH+), every drug is soluble within the bloodstream. The uncharged type (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE three.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an connected peptide of 4 to 6 amino acids.
*
FIGURE three.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with a number of layers of peptidoglycan, threaded by teichoic acids. A inset: Gram-positive envelope of Bacillus subtilis (TEM). B. Proteobacteria (Gram-negative) cells have a single layer of peptidoglycan coated by an outer membrane; the cell membrane is named the inside membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
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FIGURE three.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted within the inward-facing leaflet of the outer membrane. The C-terminal lysine varieties a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
*
FIGURE three.20 ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to 6 fatty acids. B. Repeating polysaccharide models of O antigen lengthen from lipid A.
*
FIGURE three.20a ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to 6 fatty acids.
*
FIGURE three.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide models of O antigen lengthen from lipid A.
