2. Proteins: Primary Structure Flashcards
A dipeptide can be shown as the ______ of two amino acids
In protein synthesis, the actual reactions are much more complex and involve _______ and _____
After condensation, amino acids are referred to as ______ and may contain ______ whereas Peptide groups ______
A dipeptide can be shown as the condensation of two amino acids
In protein synthesis, the actual reactions are much more complex and involve aminoacylated tRNA molecules and energy (anabolic)
After condensation, amino acids are referred to as “residues” and may contain charged sidechains, peptide groups have no charge
What is the primary structure?
- _________
- ______ connecting _____
- Potential for variation?
- ultimately* determine the _____ and ______ of polypeptides
- * other factors (eg prothtic groups, covalent modifications) will also have an influence
What is the primary structure?
- _________
- ______ connecting _____
- Potential for variation?
- ultimatelyI* determine the structure and function of polypeptides
- * other factors (eg prothtic groups, covalent modifications) will also have an influenc
Relative molecular mass or molecular weight (Mr) is the ________, and is ______
- A 10kDa compound will have an Mr of?
Relative molecular mass or molecular weight (Mr) is the mass of a compound relative to the mass of 1/12 of carbon 12, and is unitless
- A 10kDa compound will have an Mr of?
- Da = 1/12 of C12 ;
- A protein with a molecular mass of 10000g/mol = 10kDa
- 10kDa compound will have an Mr of 10000
Average molecular mass of a free amino acid is _____
Average molecular mass of an amino acid residue is _____
Average molecular mass of a free amino acid is 128 Da
Average molecular mass of an amino acid residue is 110Da
Primary structure information may include both the _____ and _____
Primary structure information may include both the sequence and disulfide bonding pattern
Insulin contains two chains produced after cleavage of a longer (86aa) polypeptide (propeptide)
Proteins w/ prosthetic groups (________ proteins) may be referred to by _________
Proteins w/ prosthetic groups (________ proteins) may be referred to by the specific prosthetic groups in questions
What are 10 types of N-terminal modifications in polypeptides?
What are three types of C-terminal modifications?
- N-Terminal modifications
- Unmodified
- Formylation (+28Da)
- Acetylation (+42Da)
- Propionylation (+56Da)
- Pyroglutamate formation
- Myristoylation; Palmitoylation
- S-Palmitoiylation
- Mono-methylation
- Di-methylation
- Tri-methylation
- C-Terminal Modifications
- Unmodified
- Methylation
- Alpha Amidation
When will proteins with different functions have different sequences?
Never, Proteins with different functions always have different sequences
Proteins with the same function typically have the same or similar sequences
Altering AA sequence often leads to change in function
What information can be found from an amino acid sequence (3)
- Structural information
- Not yet possible to predict a structure using sequence but can identify putative features/structures
- Secondary structures
- Transmembrane helices
- Signal Peptide Sequences
- Not yet possible to predict a structure using sequence but can identify putative features/structures
- Cell localization
- Info derived from comparisons w/ other sequences
- Similar sequences will generate similarities in structure and/or function
What are three types of information that can be found from sequence alignments?
- Protein function Information
- Analysis of invariant/conserved/variable amino acids in a collection of similar sequences will indicate important amino acids
- Protein Structure information
- If amino acid sequence is similar to another sequence where the structure is known, a model can be built (homology modelling)
- Evolutionary Information
- Degrees of similarity in sequences can be used to determine evolutionary history (how closely related)
Define and describe the importance of invariant residues, conservative substitutions, and non-conservative substitutions in a sequence alignment.
- Invariant residues
- Amino acids that do not vary at all between a set of sequences
- Often critical in the function of a protein
- Strengthen alignment
- Conservative Substitution
- Similar in terms of amino acid properties
- One amino acid is substituted for another with similar characteristics (2 characteristics suggest conservation)
- Non-conservative substitution
- One amino acid is substituted for another with dissimilar characteristics
- May reflect differences in functions between two proteins or less important/critical locations in structure
- *Best match for sequence alignment will have the most invariant/conserved positions
- Calculate percent identity and percent similarity in a sequence alignment.
See image
- Explain why the sequence of the same protein may be different in different organisms.
- AA sequences will change over time based on mutations
- If an amino acid mutation is non-functional, it may be detrimental (thus not retained)
- Neutral (conservative) change may be retained
- Invariant positions are more sensitive to change than others
- Degree of similarity will reflect the length of time since divergence
- Protein structure is conserved during evolution much better than protein sequence. There are numerous examples of proteins that show little sequence similarity but still adopt similar structures, contain identical or related amino acid residues in their active sites, and have similar catalytic mechanisms. These shared features support the notion that, despite low sequence similarity, such proteins are homologous
- Explain how “gaps” may appear in alignments when comparing different protein sequences and how they relate to structure.
- Two sections of aa sequences may align well but be different lengths apart
- Gaps represent insertions or deletions at the genetic level
- In protein structure, gaps typically correlate to loops on the surface
Define and distinguish between homologs, paralogs and orthologs.
-
Homologs:
- Two proteins which share a common ancestor
- Often confused with % identity
- Any two proteins which arose out of a single gene (either through duplication or speciation) can be described as homologs
-
Orthologs:
- Essentially describes the way the homologs arose
- Homologs that arose from speciation events = true homologs
- Homologs which carry out the same function in different organisms
- Essentially describes the way the homologs arose
-
Paralogs
- exist w/in the same organism; Different jobs in same process
- May have the same function or different functions as each other
›In order for evolutionary trees derived from amino acid sequences to be valid, ______ are used, not _____-
In order for evolutionary trees derived from amino acid sequences to be valid, Orthologs (speciation) are used, not _Paralogs¨(_gene duplication events
- Explain why a pure preparation of a protein is required to study its properties.
- So that we are actually studying the protein of interest rather than various contaminants
- Purification requires removal of other substances (contaminants) while maintaining structure/function of target protein
- Any given protein may have a set of protocols for purification
Protein purification is a _______ that exploits ______
Protein purification is a multistep process that exploits polypeptide characteristics¨
- Purification steps must distinguish between the protein of interest and other proteins/compounds in the preparation
- Exploit differences in:
- Solubility
- charge
- polarity
- size
- binding specificity
- Technique chosen depends on strength of difference between protein of interest and environment
- Exploit differences in:
What purification procedure are used based on the following protein characteristic?
- Solubility
- Ionic charge
- Polarity
- Size
- Binding specificity
What purification procedure are used based on the following protein characteristic?
- Solubility
- Salting out
- Ionic charge
- Ion exchange chromatography
- Electrophoresis
- Isoelectric focusing
- Polarity
- Hydrophobic interaction chromatography
- Size
- Gel filtration chromatography
- SDS-Page
- Dialysis
- Binding specificity
- Affinity Chromatography
- Explain what conditions should be controlled in a protein purification and why.
- Denaturation and degradation must be minimized during purification
- Purification protocols must take into consideration that may affect the stability/recovery of the target proteins
- pH - Buffers
- Temperatures
- ⤓Temp → ⤒Stability (T maintained at ~4-15C)
- Enzymes
- Add enzyme inhibitors in early stages
- Adsorption
- Plastic/glass may bind protein; use special/treated glassware (siliconized)
- Long term stability (oxidation, denaturation)
- cysteines oxidize to salt vapours
- Explain why a protein assay is necessary during purification, and outline three different assay techniques.
- Necessary to monitor concentration of protein of interest vs other compounds (purity)
- Three techniques:
- Directly
- Enzyme activity
- If we purified an enzyme as protein of interest
- Spectrophotometry
- Many proteins dont have unique spectrometry
- Enzyme activity
- Indirectly: measures things that interact with poi
- Antibody-linked/immunoassays
- Directly
- Define the terms activity, unit and specific activity.
-
Activity represents the ability to convert substrate into product and is indicated in units (reflection of amount)
- 1 unit = 1 micromol substrate converted per minute under defined conditions
-
Specific Activity
- The activity as a fraction of TOTAL PROTEIN
- Increases with purification as a measure of purification efficacy
- Specific activity = units/mg total protein
- Describe the criteria that determine when a protein is generally considered pure.
- Percent Recovery:
- Number of total units after a process compared to the number before that process
- Fold Purification (compare specific activity)
- Change in specific activity associated with a purification process
- If Specific activity decreases = Lost more poi than contaminant
- Change in specific activity associated with a purification process
- Describe the standard elements of a column chromatography experiment. What is the role of each standard element in a separation?
- Column Chromatography provides differential separation of compounds based on physical characteristics
- Rate of travel through column is dependent on relative affinity for either of the two phases:
-
Solid (stationary) phase
- Gel or beads held inside the column
- Affinity for this phase will SLOW migration rate through the column
-
Liquid (mobile) phase
- Fluid phase constantly flows from one end of column to the other
- Affinity for this phase will increase migration rate through the column
-
Solid (stationary) phase
- Describe how proteins are separated in ion exchange chromatography (cation and anion)
- Ion exchange chromatography
- Separation based on charge
- Charge depends on protein pI and pH of buffer
- Cation exchange:
- Stationary phase is negatively charged - more positive compounds are attracted to the beads = elute slowly
- Anion exchange:
- Stationary phase is positively charged
- More negative compounds attracted to the beads = elute slowly
- Stationary phase is positively charged
Describe how proteins are separated in size exclusion chromatography and affinity chromatography.
- Size exclusion chromatography:
- aka gel filtration chromatography
- Stationary phase consists of porous beads - openings are small enough to exclude some compounds
- Mobile phase moves faster around the beads than through them
- Small molecules will elute LATER than larger molecules
- Will not destroy quaternary structure (non-denaturing)
- Elution times compared to standard samples can yield an estimate of protein size
Describe how proteins are separated in affinity chromatography.
- Affinity chromatography
- relies on protein of interest binding to a specific ligand
- Column will contain this ligand bound (covalently) to the stationary phase
- Only proteins that bind this ligand will be retained in the column
- Addition of the ligand in solution will allow for the proteins to elute from the column (bound to free ligand rather than column )
- Describe how proteins can be modified to increase potential for affinity purification using His-tags as an example.
- Proteins may be modified to add affinity to assist in purification steps
- Ideally, engineered proteins will not affect original structure/function of protein significantly
-
His Tags:
- 6-14 Histidines inserted into polypeptide
- usually at C or N
- May increase protein solubility
- Sequential histidines can bine to nickel affinity columns
- coordination bond from imidazole ring to metal ion
- Can be eluted by changing pH or adding free imidazole
- weakens protein/column affinity
- 6-14 Histidines inserted into polypeptide
- Lowering the pH causes the metal ions to become protonated, and then they are stripped from the column.
- Describe the standard elements of a polyacrylamide gel electrophoresis (PAGE) experiment. What protein properties are exploited in a separation?
- Electrophoresis is based on the migration of charged proteins in an electric field
- The migration rate will depend on the charge/mass ratio and size
- SDS-PAGE is used to estimate the molecular weight of polypeptides
- Elements:
- Sodium dodecyl sulphate (SDS, detergent)
- binds polypeptides @ 1SDS:2AA residues
- Confers large net negative charge on the polypeptide → consistent charge-to-mass ratio
- Destroys native conformation of proteins
- SEPARATES polypeptides on basis of their mass with smaller polypeptides migrating faster through the gel matrix
- Beta-mercaptoethanol/dithiothreitol (DTT)
- Heat
- Sodium dodecyl sulphate (SDS, detergent)
- Explain how SDS binding to a protein alters protein shape and net charge.
- Sodium dodecyl sulphate (SDS, detergent)
- binds polypeptides @ 1SDS:2AA residues
- Confers large net negative charge on the polypeptide → consistent charge-to-mass ratio
- Destroys native conformation of proteins
- SEPARATES polypeptides on basis of their mass with smaller polypeptides migrating faster through the gel matrix
- Explain why a dye such as Coomassie Blue is used after electrophoresis.
- Coomassie blue binds to PROTEINS not the gel, enabling each polypeptide to be seen as a discrete band
- Smaller polypeptides migrate faster through the gel and are found closer to the bottom
- Describe how protein or subunit molecular weight is determined by SDS-PAGE.
- Log Mr (relative molecular mass) vs migration distance will be linear over a certain range
- Can be used to estimate molecular weight
- Quaternary structure is destroyed
Describe additional information that may be obtained from 2-D electrophoresis compared to SDS-PAGE or IEF alone.
Two-dimensional electrophoresis is a powerful technique that combines two different electrophoresis techniques, separating molecules by two different properties, resulting in a greater resolving power than each technique alone. For example, IEF in the first dimension and SDS-PAGE in the second dimension will separate proteins according to charge, followed by molecular mass.
Outline core principle in protein purification that influence specific choices in a particular step.
different proteins have different amino acid sequences and spatial structures, leading to differences in their physical, chemical, and biological properties
Purification steps must distinguish between the protein of interest and other proteins/compounds in the preparation.
Draw the chemical reaction of peptide bond formation.
- Amino Acids bind to each other to make polypeptide chains via a condensation reaction, which involves the elimination of a water molecule for each bond formed
- See image: 2 amino acids with the carboxylic acid group of the first amino acid next to the amino group of the second amino acid
- The hydroxyl group of amino 1 and one of the hydrogen atoms in the amino group of amino acid two
- These will be eliminated as water
- Two amino acids joined together with the carboxylic C from amino acid 1 single-bonded to the N from amino acid 2
Expand?? unclear
Explore the variety of sizes and combinations of biologically active peptides and proteins.
- (insulin and leupeptin
- Insulin:
- ~5600Da
- (21+30)aa x 110Da/aa = 5610
- Actual 5808
- Contains 2 chains produced after cleavage of a longer chain (86aa) propeptide
- ~5600Da
- Leupeptin
- tripeptide that acts as an inhibitor for some proteases
What is the sequence of the peptide?
Identify the peptide
Identify any post translation modifications
N-formyl methionine is part of protein synthesis initiation for bacteria and appears (transiently) at the N-terminal end of the protein. What would be the impact of this residue being present?
A.The N-terminus would be positively-charged.
B.The N-terminus would be neutral.
C.The N-terminus would be negatively-charged
B. The N-terminus would be neutral
Estimate molecular weight from polypeptide length (and vice versa).
eg insulin:
~5600 Da (insulin)
Alpha chain = 21 aa
Beta chain = 30aa
(21+30) x 110aa/Da = 5600
(Average molecular mass of an amino acid residue is 110 Da)
Draw the structures of modified amino acid termini, including
- methylation *C-Terminal*,
- amidation,
- acetylation
- acylation
ester on protein
Draw the structures of modified amino acid termini, including
- methylation N-Terminal
- amidation,
- acetylation
- acylation
Draw the structures of modified amino acid termini, including
- methylation,
- amidation C-Terminal
- acetylation
- acylation
Draw the structures of modified amino acid termini, including
- methylation,
- amidation,
- acetylation: N-terminal
- acylation
Name and describe 6 post translational modifications in image
- Disulfide cross-linking
- Occurs between two cysteine residues
- Hydroxylation
- Addition of hydroxyl group
- Carboxylation
- Addition of carboxyl group
- Phosphorylation
- Addition of phosphate group
- Methylation
- Addition of methyl group
- Acetylation
- Addition of acetyl group
Pending
Draw the structures of modified amino acid termini, including
- methylation,
- amidation,
- acetylation: N-terminal
- acylation
?
