Most proteins have at least one what?
Trp (Tryptophan, W)
Trp absorbs UV light at ____ nm.
280nm only!
Trp is the only amino acid that does what?
Blocks light
_______ can be determined based on the detection of Trp in proteins.
Protein
Every protein begins with a _______ amino acid.
Methionine, tells you where to start reading.
Cys residues can form what?
Disulfide bonds
Disulfide bonds can form what?
Intra-strand crosslinks
Disulfide bonds facilitate what?
Crosslinking
When a protein contains more than one ________, disulfide bonds can also form __________.
Polypeptide chain; Inter-chain crosslinks.
Which amino acid attributes the most protein absorbance at 280 nm?
Tryptophan
Every protein starts with the amino acid______?
Methionine
Disulfide bond is formed through the side chain of which amino acid?
Cysteine
What makes proline so unique from all other amino acids?
It is the only one with a side chain that wraps back onto itself which causes the rigidity within the molecule that cause kink in the peptide wherever there is a proline.
Amino acids are linked via a what?
Condensation reaction (where a water molecule is eliminated). The polymerization of amino acids to form a polypeptide chain involves the condensation of the carboxylate group of one amino acid with the amino acid group of another.
Amino acids are linked by what?
Peptide bonds
Amino acids are linked by peptide bonds to form a?
Polypeptide
Polypeptides have many amino acid ____?
Residues, which are amino acids within a peptide because only the residual atoms remain.
A chain of amino acid residues linked by peptide bonds is written or drawn so that the residue with a free amino group is on the ______, and the residue with a free carboxylate group is on the _____.
Left (this end of the polypeptide is called the N-terminus); Right (this end is called the C-terminus).
T/F The terms "protein" and "polypeptide are fully interchangeable.
False, protein is a general term.
What is a protein's primary structure?
The sequence of amino acids in a polypeptide.
What type of covalent bonds are responsible for holding together the primary structure of proteins?
Peptide or amide bonds.
The secondary structure is?
The localized conformation of the polypeptide backbone.
What are two kinds of secondary structures?
Alpha(a) helix and Beta (B) sheets.
What causes the secondary structure to form?
The formation of secondary structure is dictated by the steric hindrance of the peptide bond and the side chains.
In a Alpha helix structure, the polypeptide backbone twists in a ________ helix.
Right-handed helix (DNA helix is also right-handed)
The Alpha helix structure is stabilized by the ____ bonds formed between the _____ oxygen and the ______ hydrogen.
H- ; Carbonyl ; Amino
A complete turn of a Alpha helix is made up of _____ amino acids and raise the helix by ____ A.
3.6 ; 5.4
Most Alpha helices are about ___ residues long and the side chains extend ______ from the helix.
12 ; Outward
The Beta sheet is ____ in structure.
Planar
Like the Alpha helix, the Beta sheet is also stabilized by the ___ bonds formed between the ______ oxygen and the ______ hydrogen.
H- ; Carbonyl ; Amino
What are the two types of Beta sheets?
Parallel B sheets and Antiparallel B sheets.
Parallel beta sheets
Neighboring chains run in the same direction.
Antiparallel beta sheets
Neighboring chains run opposite directions. (More relaxed state)
Alpha helix and Beta sheets are classified at regular secondary structures because?
Their component residues exhibit backbone conformations that are the same from one residue to the next.
In every protein, elements of secondary structure are linked together by ______ of various sizes.
Polypeptide loops
The loops that link B strands or Alpha helices consist of residues with what?
Irregular secondary structures
Irregular secondary structures
The polypeptide does not adopt a defined secondary structure in which successive residues have the same backbone conformation. They are mostly found in the linker region connecting the Alpha helices and Beta strands.
The tertiary structure
The over all three-dimensional SHAPE of the protein made up of the secondary structures.
What is a domain?
A polypeptide segment that has folded into a single structure unit with a hydrophobic core.
What are the leading factors that hold tertiary structures together?
Hydrophobic forces and cross linkers.
Protein folding and protein stabilization depend on _______ forces.
Noncovalent
The location of a particular side chain in a protein's tertiary structure is related to its what?
Hydrophobicity, which is the greater a residue's hydrophobicity, the more likely it is to be located in the protein interior.
Side chains pack together in the protein _____, leaving very little empty space or space that could be occupied by a water molecule.
Interior
A folded polypeptide assumes a shape with a ________ surface and a ________ core.
Hydrophilic; Hydrophobic
Which amino acid would most likely be found in the internal core of a globular protein? Lys, Leu, Glu, Ser
Leu, because Leucine is hydrophobic.
Protein structures are stabilized mainly by what?
The hydrophobic effect
Many folded polypeptides appear to be held in place by what?
Cross-links
Cross-links help?
Stabilize the protein
Cross-linkers found in proteins
Ion pair linkers, disulfide bond linkers, and metal linkers.
Ion pair linkers
Form between oppositely charged side chains or the N- and C- terminal groups. The electrostatic interaction is strong but does not contribute much to protein stability.
Disulfide bond linkers
Form within and between polypeptide chains.
Protein folding begins with the formation of _________ structures.
Secondary
All the information required for a protein to fold is contained?
In its amino acid sequence
The quaternary structure refers to what?
The number of polypeptides in a protein.
The individual chains are called?
Subunits
If the subunits are all identical it is known as what?
Either a homodimer, homotetramer, etc.
If the subunits are not all identical, what prefix is used?
Hetero
Domains vs. subunits
A single chain can form a local SD structure domains. Two or more separate chains (subunits) can orient in 3D space to give quaternary structure.
A protein that consists of a single polypeptide chain will lack which level of protein structure?
Quaternary, they contain two or more separate chains or subunits.
What type of covalent bonds are responsible for holding together the tertiary structure of proteins?
Hydrophobic forces and cross linkers.
What is Chromatography?
A technique for separating molecules on the basis of size, charge, or specific binding. Proteins or other solutes pass through the column at different rates, depending on how they interact with the stationary phase.
Gel filtration (or size exclusion) chromatography
Used to separate protein based on its fluid dynamics. Larger proteins will be excluded faster than smaller proteins which will spend time inside the beads.
Size exclusion chromatography separates proteins based on what?
Their hydrodynamic volume.
In size exclusion, as proteins pushed through the column by the flow of the buffer, the ______ proteins take a more direct route while the _____ ones take a more indirect route.
bigger; smaller
In size exclusion, the cause of separation of protein is based on their hydrodynamic ______ or ______. This is why bigger proteins come out faster and the smaller proteins come out slower.
Volume; natural size.
What is the limitation of size exclusion chromatography?
It can not breakup the protein's quaternary structure.
SDS PAGE (Poly-acrylamide Gel Electrophoresis) is used to?
Analyze protein by its peptide size.
In SDS PAGE
Both the sample and the gel contain the detergent SDS which binds to proteins to give them a uniform density of negative charges.
SDS PAGE separates proteins based on?
Size in their denatured state.
The mechanism used in SDS PAGE is?
The electromagnetic poly-acylamide gel.
In SDS PAGE it is easier for the _____ proteins to go through the gel than it is for the _____ protein. So bigger proteins are on the top and smaller proteins are on the bottom.
Smaller; Bigger
What is the limitation of SDS PAGE?
It can not purify proteins, it only analyzes them. The protein is destroyed (denatured) in the process.
Ion exchange chromatography is used when?
Separating protein by their charge.
Resins (the solid phase) in Ion exchange have either _____ or _____ functional groups.
Diethylaminoethane (DEAE) (positive charged) or Carboxymethyl (CM) (negative charged).
In Ion exchange, negatively charged proteins will bind tightly to the _____ groups, while uncharged and positively charged proteins pass through the.
Diethylaminoethane (positive charged) groups
Positively charged columns grab anything _____ charged and is called _____ columns.
Negatively; Anion
Negatively charged columns grab anything _____ charged and is called _____ columns.
Positively charged; Cation
What are limitations of Ion exchange?
It can not purify proteins that are pH sensitive. This is because often times sample buffer pH has to be either lowered or increased before being loaded to the column.
Which of the following separates proteins based on net charge? Size-exclusion chromatography, Ion exchange chromatography, or SDS PAGE
Ion exchange chromatography
Which of the following separates protein based on their hydrodynamic volume?
Size-exclusion chromatography.
What is the definition of isoelectric point(PI)?
The exact pH at which the overall charge of a compound is zero.
What are two common techniques to study the protein structure?
X-ray crystallography and Nuclear magnetic resonance (NMR)
X-ray crystallography
Is performed on samples of protein that have been induced to form crystals.
Pros of x-ray crystallography
High solution, sees every atom, and can study large protein structures.
Cons of x-ray crystallography
Can only study protein in static condition or crystal form, and it is technically difficult.
Nuclear magnetic resonance (NMR)
Takes advantage of the ability of atomic nuclei (most commonly hydrogen) to resonate in an applied magnetic field according to their interactions with nearby atoms.
Pros of NMR
Can study the movements of proteins and can study protein in solution.
Cons of NMR
Low resolution, can not study big protein structure, and can only see hydrogen and nitrogen isotope atoms.
Which technique produces higher resolution data? X-ray crystallography or NMR
X-ray crystallography
Which technique can be used to study protein dynamics? X-ray crystallography or NMR
NMR
Compare and contrast the structures of DNA helix and the Alpha helix.
Both turn in the right-handed direction. Both have tightly packed interiors: In DNA the interior of the helix is occupied by nitrogenous bases and in the Alpha helix, the atoms of the polypeptide backbone contact one another. In Alpha helix, the side chai
How to calculate pI formula
pI= 1/2 (pK1 + pK2)
What are the techniques that separate and analyze proteins?
Gel filtration (or size exclusion) chromatography, Ion exchange chromatography, Affinity chromatography, and SDS PAGE.
What are common techniques used to study the protein structure?
X-ray crystallography and NMR.