Protein Analysis Microbiology
Escherichia coli: a Gram-negative bacterium of the gut microbiome, Part 1
FIGURE 3.1 ■ Escherichia coli: a Gram-negative bacterium of the gut microbiome. The envelope includes the outer membrane; the cell wall and periplasm; and the inner (cell) membrane. Embedded in the membranes is the motor of a flagellum. The cytoplasm includes enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, which are folded by chaperones. The nucleoid contains the chromosomal DNA wrapped around binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
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Bacterial Cell Structure: What is seen in Gram-negative & Gram-positive bacteria
Bacteria can be placed in 2 groups based on the thickness and placement of the cell wall
Gram-negative
Gram-positive
Plasma membrane
Absence of a nucleus
DNA is located in the nucleiod region
No histone proteins, but DNA-binding proteins present to keep genomic DNA compact
Plasmids: DNA that is independent of the genome.
Flagellum
Biochemical composition of bacteria
Water
Essential Ions
Needed for enzymatic reactions
Small organic molecules: lipids and sugars
Lipids are almost as abundant as RNA molecules
Found in the cell wall
peptidoglycan
Macromolecules: nucleic acids, proteins, fats, & sugars
Goal: Isolate proteins
Purpose of cell fractionation is to isolate components of choice from a bacterial cell
The first step is cell lysis
EDTA
Sucrose
Lyzozymes
Ultracentrifugation
FIGURE 3.2 ■ 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 inner and outer membranes. Photo
Source: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
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Goal 2:Protein Analysis
FIGURE 3.3 ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation. B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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FIGURE 3.3a ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation.
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FIGURE 3.3b ■ Protein analysis.
B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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Understanding the role of a protein
FIGURE 3.4 ■ Genetic analysis of FtsZ.
A. E. coli with aspartate (D) at position 45 replaced by alanine (A) (D45A) elongate abnormally, forming blebs from the side, with no Z-rings. Cells with aspartate replaced by alanine at position 212 (D212A) elongate to form extended nondividing cells that contain spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Model of FtsZ protein monomer based on X-ray crystallography shows the position of the mutant residues, D212A and D45A.
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FIGURE 3.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids. Half the membrane volume consists of proteins.
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LeuT sodium/leucine cotransporter
Homology to human neurotransmitter sodium sympoters
Has been used as a blueprint to understand structure and function, and pharmacology of NSS human transporters.
FIGURE 3.7 (part 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex bacteria.
The protein complex carries leucine across the cell membrane into the cytoplasm, coupled to sodium ion influx. (PDB code: 3F3E)
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Transport across bacterial membranes
Passive diffusion
Membrane proteins
Aquaporins
Permease (lac operon)
Osmosis
Greater osmotic pressure can lead to bacterial cell lysis (seen with certain antibiotics)
Membrane-permeant weak acids and bases: can cross the plasma membrane
Transmembrane ion gradients
FIGURE 3.8 ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
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FIGURE 3.8a ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
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FIGURE 3.8b ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
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NAM and NAG are linked together by a β-(1,4)-glycosidic bond
Lysozymes target this bond
The peptidoglycan monomer will have 5 peptides
Once this monomer becomes incorporated into the existing polymer, 4 peptides are seen.
FIGURE 3.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an attached peptide of four to six amino acids.
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FIGURE 3.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with multiple 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 covered by an outer membrane; the cell membrane is called the inner membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
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Gram +
FIGURE 3.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
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FIGURE 3.20 ■ 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 six fatty acids. B. Repeating polysaccharide units of O antigen extend from lipid A.
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FIGURE 3.20a ■ 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 six fatty acids.
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FIGURE 3.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide units of O antigen extend from lipid A.
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FIGURE 3.1 ■ Escherichia coli: a Gram-negative bacterium of the gut microbiome. The envelope includes the outer membrane; the cell wall and periplasm; and the inner (cell) membrane. Embedded in the membranes is the motor of a flagellum. The cytoplasm includes enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, which are folded by chaperones. The nucleoid contains the chromosomal DNA wrapped around binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
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FIGURE 3.2 ■ 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 inner and outer membranes. Photo
Source: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
*
FIGURE 3.3 ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation. B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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FIGURE 3.3a ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation.
*
FIGURE 3.3b ■ Protein analysis.
B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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FIGURE 3.4 ■ Genetic analysis of FtsZ.
A. E. coli with aspartate (D) at position 45 replaced by alanine (A) (D45A) elongate abnormally, forming blebs from the side, with no Z-rings. Cells with aspartate replaced by alanine at position 212 (D212A) elongate to form extended nondividing cells that contain spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Model of FtsZ protein monomer based on X-ray crystallography shows the position of the mutant residues, D212A and D45A.
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FIGURE 3.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids. Half the membrane volume consists of proteins.
*
FIGURE 3.7 (part 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex bacteria.
The protein complex carries leucine across the cell membrane into the cytoplasm, coupled to sodium ion influx. (PDB code: 3F3E)
*
FIGURE 3.8 ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.8a ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.8b ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an attached peptide of four to six amino acids.
*
FIGURE 3.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with multiple 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 covered by an outer membrane; the cell membrane is called the inner membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
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FIGURE 3.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
*
FIGURE 3.20 ■ 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 six fatty acids. B. Repeating polysaccharide units of O antigen extend from lipid A.
*
FIGURE 3.20a ■ 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 six fatty acids.
*
FIGURE 3.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide units of O antigen extend from lipid A.
