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biochemistry chapter 5 - Proteins: Their Primary Structure and Biological Function
Terms in this set (93)
Proteins Fall into Three Basic Classes According to Shape and Solubility
- protein with simple, regular linear structures
- serve structural roles in cells
- insoluble in water or in dilute salt solutions
- a protein roughly spherical in shape
- The polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior
of the molecule and the hydrophilic side chains are on the outside exposed to the solvent, water
- very soluble in aqueous solutions.
ex. cytosolic enzymes
- protein that are associated with the various membrane
systems of cells
- have hydrophobic amino acid side chains that are oriented towards the nonpolar phase within membrane
- insoluble in aqueous solutions but can be solubilized in solutions of detergents
- have very few hydrophilic amino acids
Protein Structure Is Described in Terms of Four Levels of Organization
1. Primary structure (1°) - polypeptide sequence
2. Secondary structure (2°) - local structures - H-bonds (alpha helix and beta pleated sheets)
3. Tertiary structure (3°) - overall 3-dimensional shape (ionic, disulfide bridges, H bonds, noncovalent weak intermolecular forces)
4. Quaternary structure (4°) - subunit organization (multiple tertiary structures interaction)
The amino acid sequence of a polypeptide
- peptide bonds
hydrogen-bonding interactions between adjacent
amino acid residues
1. alpha helix
2. beta pleated sheets
When ONE polypeptide chain of protein molecules bend and fold in order to assume a more compact three-dimensional shape
- form a globular shape
- noncovalent forces such as hydrogen bonds
and ionic, van der Waals, and hydrophobic interactions, disulfide bonds
composed of compactly folded polypeptide chains arranged in a spherical form
- hydrophobic in the middle and hydrophilic on the outer
interaction between two different tertiary proteins
noncovalent forces such as hydrogen bonds
and ionic, van der Waals, and hydrophobic interactions and disulfide bonds
What is a protein's conformation?
- 3-D structure of a protein
- A protein, or any molecule, can change its conformation by changing shape without breaking a bond.
How can a protein change its conformation?
through the rotations about each of the single bonds in the amino acids
What is a protein's configuration?
- D or L orientation
- geometric possibilities for a particular set of atoms
- Rearrangements between configurational alternatives can be achieved only by breaking and remaking bonds.
Which protein property can be changed without breaking bonds? conformation or configuration ?
Which protein property CANNOT be changed without breaking bonds? conformation or configuration ?
How are proteins separated? 2 characteristics
2. electrical charge
strategies that take advantage of the biological function
or specific recognition properties of a protein
When are proteins least soluble?
1. at their isoelectric point
- the molecule is neutral charge and unlikely to be soluble in water (no H bond interactions)
- electrostatic repulsion between protein molecules is minimal
- more likely to precipitate out of solution
2. high salt concentrations
- the pH value at which the sum of their positive and negative electrical charges is zero
protein analysis methods
1. UV-VIS spectroscopy
3. BCA reagent, Bradford reagent
4. SDS-PAGE electrophoresis
5. Isoelectric focusing
6. 2D gel electrophoresis
7. mass spectrometry
Protein purification methods
1. Dialysis - buffer exchange, clean-up
2. Ion exchange chromatography - charge
3. Gel filtration - size
4. Affinity chromatography - affinity to a small molecule
5. Hydrophobic Interaction chromatography
mechanism of salting in - in terms of protein solubility
- when ion concentrations is low protein solubility is high due to the interaction between ions and protein that reduce the electrostatic interactions between proteins
mechanism of salting out - in terms of protein solubility
- when ion concentration is high protein solubility is low due to the ions outcompeting the protein in solvation
- water is more attracted to the ions then the protein which causes the proteins to interact which each other and precipitate out of solution
What is dialysis?
- a type of Protein purification method which is used to separate protein from small molecules in solution by diffusion across a semipermeable membrane (based on size and concentrations)
- since protein are much bigger then small molecules such as salts, reducing agents, or dyes they will remain the bag while the small molecules diffuse out into the outer low concentration solution
1. The solution of macromolecules (mixture of different protein and solutes) is placed in a semipermeable membrane bag
2. the bag is immersed in a buffer with a lower solute concentration than what is in the bag.
3. Diffusible solutes in the dialysis bag will equilibrate across the membrane. (high to low con. movement out of the bag)
4. since protein molecules are large they cannot diffuse across the membrane
Ion Exchange Chromatography
- a type of Protein purification method which is used to Separate Molecules on the Basis of Charge
- desired + or - protein in solution are attracted to the + or - charged resin beads in a column and all other neutral or oppositely charged molecules will flow through the column
- Cation exchange chromatography is used when the desired molecules to separate are cations (the resin beads are negatively charged)
- anion exchange chromatography is used to separate anions ( the beads in the column are positively charged)
is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte.
Ion Exchange Chromatography procedure
1. solution with protein mixture is poured into a column with resin beads that have a specific charge (negative or positive) depending on the charged of the desired protein
2. if desired protein is negatively charged the resin will be positively charged. Negatively charged ions will attract to the resin and allow the neutral or positively charged protein to flow through
Gel Filtration Chromatography
- a type of Protein purification method
- Can Be Used to Separate Molecules on the Basis of Size
- large porous beads in a column will trap small molecules but larger molecules such as protein are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- a Protein analysis technique that can Be Used to Separate protein on the basis of size and not charge
- a negatively charged surfactant called SDS is used to bind to the protein to make the net charge negative so that charge does not affect migration
- protein sample is loaded into a polyacrylamide porous gel contained in a pH buffered solution with a constant voltage applied which causes the movement of proteins through the gel from negative to positive electrode
- the rate of migration of a protein is proportional to molecular weight
what does SDS do?
-is a negatively charged surfactant that binds polypeptides in a quantity directly proportional to the length of the polypeptide
-makes every aa negatively charged
**allow the polypeptide to move along electric gradient based on molecular weight ONLY
Two-Dimensional Gel Electrophoresis
- a type of Protein purification method
Macromolecules are first separated according to charge by isoelectric focusing in a tube gel. The gel containing separated molecules is then place on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS-PAGE gel, where they are separated according to size.
1. isoelectric focusing
- a type of Protein purification method
-is a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand
Affinity Chromatography procedure
1. Small-molecule targets (ligands) are immobilized through covalent attachment to an insoluble matrix, such as cellulose or polyacrylamide, in a column.
2. The protein of interest is then passed through the column, where it binds to the target ligands, while other proteins pass through without binding.
3. The protein can be eluted by addition of high concentrations of the free ligand.
- a protein analysis method that allows one to determine the presence and concentration of aromatic amino acids because they are the only amino acids that have an absorbance in the ultraviolet-visible spectral region (280nm)
3 aromatic aa
- is a technique for separating different molecules by differences in their isoelectric point (pI) as they move through immobilized pH gradient (IPG) gels while a constant voltage is applied
- proteins will stop moving once they reach their isoelectric point (their net charge is zero)
BCA reagent / smith assay
- a protein analysis method used to determine the presence and total concentration of protein in a solution through a reaction between protein and BCA which produces a color change from green to purple which is proportional to protein concentration
Bradford reagent / Bradford protein assay
- a protein analysis method
used for determining the presence of protein that is based on the reaction of the dye Coomassie Brilliant Blue G-250 with the protein and changes color from brown to blue
*** The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.
mass spectrometry in protein analysis
- is an analytical technique that can be used to determine amino acid sequence of protein through ionization into charged fragments which are then sorted based on their mass to charge ratio and their abundance measured which creates a spectra of chemical fragments of the original protein
1. evaporate and ionize molecules in a vacuum, creating gas-phase ions
2. separate the ions in space and/or time based on their m/z ratios
3. measure the amount of ions with specific m/z ratios
In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with electrons. This may cause some of the sample's molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.
Hydrophobic Interaction chromatography
Which of the following methods uses the absorbance of aromatic residues in proteins to determine the concentration of the protein?
All of the above
Which of the following methods separate proteins according to their charge?
Ion exchange chromatography
b and c
b and c
How is a polypeptide chain read?
N-terminal end of the polypeptide chain through to the
Who was the first scientist to determine the Sequence of a Protein?
Frederick Sanger sequenced the amino acids in insulin
Proteins can be sequenced in two ways
1. real amino acid sequencing
2. sequencing the corresponding DNA in the gene for the protein
Determining the amino acid Sequence of a protein - A Six-Step Strategy
1. If more than one polypeptide chain, the chains are separated and purified.
2. Intrachain S-S (disulfide) cross-bridges are cleaved.
3. The N-terminal and C-terminal residues are identified
4. Each polypeptide chain is cleaved into smaller fragments, and the composition and sequence of each fragment is determined.
5. Step 4 is repeated, using a different cleavage procedure to generate a different and overlapping set of peptide fragments.
6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments.
1. separate polypeptide chains from each other = disrupt weak noncovalent interactions
2. break disulfide bonds
3. determine N and C terminus
4. break peptide chain into fragments with an enzyme
5. repeat fragmentation of protein using different enzymes
5. overlap fragments and determine original sequence
step 1 of amino acid sequence identification
- Separation of heteromultimeric chains which are held together by weak intermolecular forces (H bonds, ionic bonds, hydrophobic interactions, van der wals)
Separation is achieved with:
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually ammonium sulfate)
step 2 of amino acid sequence identification
- Cleavage of Disulfide bridges
1. Performic acid oxidation (break S-S bond and prevent reformation)
2. Sulfhydryl reducing agents (break S-S bond)
- dithiothreitol (DTT) or dithioerythritol
SH modification (prevent S-S reformation)
- iodoacetate (alkylating agent)
- 3-bromopropylamine (modification)
step 3 of amino acid sequence identification
- Identify the amino acids at the N- and C-terminal of the polypeptide chain
N-terminal amino acid identification:
- Edman's reagent = phenylisothiocyanate = PTH label the N-amino acid and make an amino-PTH derivative
- Enzymatic analysis (carboxypeptidase)
Carboxypeptidase A - cleaves any residue except Pro, Arg, and Lys
Carboxypeptidase B (hog pancreas) only works on Arg and Lys
How to identify the N-terminus amino acid and sequential amino acids?
- is a method of sequencing amino acids in a peptide in a sequential order starting from the N-terminus
- the Edman reagent binds to the free N-terminus of a polypeptide (labelling)
- the N terminus amino acid can then be excised and recovered as a PTH derivative (ex. ala-PTH)
- the rest of the polypeptide remains intact and undisturbed
- use Chromatography to identify the PTH derivative
- Repeat multiple cycles to identify the sequence of the polypeptide
What is the Edman reagent?
phenylisothiocyanate (phenyl-N=C=S) = PTH ---> combines with the free amino terminus of a protein
How to identify the C-terminus amino acid and sequential amino acids?
are enzymes that cleave amino acid residues from the C-terminus of polypeptides in a successive fashion
Four carboxypeptidases used to identify C-terminus of polypeptides
1. Carboxypeptidase A (from bovine pancreas) - hydrolyzes the C-terminal peptide bond of all residues except proline, arginine, and lysine. (NOT PRK)
2. Carboxypeptidase B (from hog pancreas) - is effective only when Arg or Lys are the C-terminal residues.
3. Carboxypeptidase C (from citrus leaves) - cleaves ANY C-terminus residue
4. Carboxypeptidase Y (from yeast) - cleaves ANY C-terminus residue
step 4 and 5 of amino acid sequence identification
- Fragmentation of the chains
1. Enzymatic fragmentation
D. Staphylococcal protease
E. endopeptidase Lys-C
2. Chemical fragmentation
A. cyanogen bromide (CNBr)
major proteolytic enzymes
D. Staphylococcal protease
E. endopeptidase Lys-C
a protease that cleaves ONLY on the C-side of Arg or Lys, generating a set of peptide fragments having Arg or Lys at their C-terminus
N-ala-tys-cys-arg- (CLEVAGE SITE)-phe-met-C
N-ala-tys-cys-arg-C + N-phe-met-C
an enzyme that breaks down proteins and peptides.
the breakdown of proteins or peptides into amino acids by the action of enzymes
a protease that USUALLY cleaves on the C-side of aromatic amino acids : trp, phe and tyr
***not very specific - can cleave Leu sometimes too
is a protease that ONLY cleaves on the C side of arginine
N-ala-tys-cys-arg- (CLEVAGE SITE)-phe-met-C
N-ala-tys-cys-arg-C + N-phe-met-C
a protease that cleaves ONLY on the C-side of asp or glu, generating a set of peptide fragments having asp or glu at their C-terminus
is a protease that ONLY cleaves on the C side of lysine
Cyanogen Bromide (CNBr) chemical proteolysis
- chemical methods of proteolysis that ONLY cleaves the C-side of met
- Be able to recognize the results = a peptide with a C-terminal homoserine lactone
- The reaction of cyanogen bromide (CNBr) with a peptide results in cleavage at Met residues and produces peptides with C-terminal homoserine lactone residues where Met residues once were.
Cyanogen Bromide cleavage of met from polypeptide procedure
1. Cyanogen bromide (CNBr) is a highly selective reagent for cleavage of peptides only at methionine residues.
Step 1: Nucleophilic attack of the Met S atom on the -CN carbon atom, with displacement of Br.
Step 2: Nucleophilic attack by the Met carbonyl oxygen atom on the R group. The cyclic derivative is unstable in aqueous solution.
Step 3: Hydrolysis cleaves the Met peptide bond. C-terminal homoserine lactone residues occur where Met residues once were.
step 6 of amino acid sequence identification
- Reconstructing the sequence
1. Use two or more fragmentation agents in separate fragmentation experiments
2. Sequence all the small peptides produced (usually by Edman degradation)
3. Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain
Homologous Proteins from Different Organisms Have
Homologous Amino Acid Sequences
Proteins sharing a significant degree of sequence similarity and structural resemblance are said to be
Proteins that perform the same function in different
organisms are also referred to as
- are proteins from DIFFERENT species that have
homologous amino acid sequences (and often a similar function)
source: common ancestral gene during evolution ( there was speciation)
- are proteins found within a SINGLE species that have homologous amino acid sequences
source: gene duplication
BLAST (Basic Local Alignment Search Tool)
is one commonly used program for rapid searching of DNA sequence databases to find matches. The
program detects local as well as global alignments where sequences are in close agreement. Even regions of similarity shared between otherwise unrelated proteins
can be detected. Discovery of sequence similarities between proteins can be an important clue to the function of uncharacterized proteins. Similarities are also useful
in assigning related proteins to protein families.
- The optimal sequence alignment between two proteins is one that maximizes sequence alignments while minimizing gaps.
BLOSUM is an acronym for Blocks Substitution
a matrix that scores each position on the basis of observed frequencies of different amino acid substitutions within blocks of local alignments in related proteins.
Phylogeny of Cytochrome c
- The number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived
- This observation can be used to build phylogenetic trees of proteins
- This is the basis for studies of molecular evolution
Can Polypeptides Be Synthesized in the Laboratory?
- Chemical synthesis of peptides and polypeptides of defined sequence can be carried out in a laboratory.
- Strategies are complex because of the need to control side chain reactions
- Blocking groups must be added and later removed
- du Vigneaud's synthesis of oxytocin in 1953 was a milestone
- Bruce Merrifield's solid phase method was even more significant
Solid Phase Synthesis of Peptides
- R. Bruce Merrifield and his collaborators pioneered the solid-phase synthesis of polypeptides in the laboratory
1. Carboxy terminus of a nascent peptide is covalently anchored to an insoluble resin
2. After each addition of a residue, the resin particles are collected by filtration (waste, byproducts - removed)
- proteins that consist of only amino acids and contain no other chemical groups
- Proteins may be "conjugated" with other chemical groups
- covalent modification of amino acid side chains in proteins after the protein has been synthesized
If the non-amino acid part of the protein is important to its function
any of a class of proteins that have carbohydrate groups attached to the polypeptide chain
any of a group of soluble proteins that combine with and transport fat or other lipids in the blood plasma
a complex consisting of a nucleic acid bonded to a protein.
a protein that contains phosphorus (other than in an associated nucleic acid or phospholipid).
is a generic term for a protein that contains a metal ion cofactor
metalloprotein (iron) containing a heme prosthetic group- an organic compound that allows a protein to carry out several functions that it cannot do alone. Heme remains bound to the protein permanently, either covalently or noncovalently bound or both
are proteins that contain a nucleic acid derivative of riboflavin: the flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN)
most common post-transnational modifications
1. cleavage of N-signal sequence - that stipulate protein proper destination
** MEMORIZE THE CHART
The general term for proteins containing nonprotein constituents
is the complete catalog of proteins encoded by a genome; in cell-specific terms
- is the complete set of proteins found in a particular cell type at a particular time
- the unified atomic mass unit symbol
- is the standard unit that is used for indicating mass on an atomic or molecular scale (atomic mass). One unified atomic mass unit is approximately the mass of one nucleon (either a single proton or neutron) and is numerically equivalent to 1 g/mol
1 Da = 1 g/mol
* MUST KNOW FOR MOLECULAR WEIGHT CALCULATIONS FOR PEPTIDE SEQUENCING
What is the average mass of 1 amino acid?
is the addition of hydrophobic molecules to a protein or chemical compound
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