Term
| H bond donors vs acceptors |
|
Definition
Donors - have a proton bound to an EN atom (N, O, F)
Acceptors - have a LP of electrons |
|
|
Term
| Which functional groups can be donors and acceptors for H-bonds? |
|
Definition
|
|
Term
|
Definition
An entropic effect, NOT a force; see the entropic release of water molecules during aggregation of non-polar solutes in solution
Primary reason and contribution to protein conformational changes in folding |
|
|
Term
If pH = pKa -1
If pH = pKa + 1 |
|
Definition
Higher concentration of protonated form -> 90% HA, 10% A-
Higher concentration of deprotonated form -> 10% HA, 90% A- |
|
|
Term
| What is the only non-chiral amino acid? |
|
Definition
|
|
Term
| Which aa's are S amino acids, and which are R |
|
Definition
19 of the amino acids are S, as well as L
Cys is R because the -SH group changes priority (also an L-amino acid though) |
|
|
Term
| Non-Polar Aliphatic Amino Acids |
|
Definition
| Glycine, Alanine, Proline, Valine, Leucine, Isoleucine, Methionine |
|
|
Term
|
Definition
| Phenylalanine, Tyrosine, Tryptophan |
|
|
Term
| Neutral, Polar Amino Acids |
|
Definition
| Serine, Threonine, Cysteine, Asparagine, Glutamine |
|
|
Term
| Neutral, Polar Amino Acids |
|
Definition
| Serine, Threonine, Cysteine, Asparagine, Glutamine |
|
|
Term
| Polar, Positive Amino Acids |
|
Definition
| Lysine (10.5), Arginine (12.5), Histidine (6) |
|
|
Term
| Polar, Negative Amino Acids |
|
Definition
| Aspartate, Glutamate (both are 4.0) |
|
|
Term
|
Definition
| The pH at which a molecule has NO net charge (can take the average of the two closes pK values of the molecule) |
|
|
Term
|
Definition
Experimentally measures pI; type of electrophoresis
Create pH gradient & run electric field through gel - unnknown molecule will move until it reaches its pI in the gradient |
|
|
Term
|
Definition
| SDS = detergent; binds to hydrophobic regions of proteins and causes proteins to unfold (all proteins have same shape); binds based on side (all proteins with the same charge/mass ratio) -> so separation of proteins based on MOLECULAR WEIGHT |
|
|
Term
| What is the amount of dye that binds proportional to? |
|
Definition
| Proportional to the MW of the protein (mass); also need to consider the mole ratios of the protein |
|
|
Term
| In what conditions are S-S bonds formed/broken under? |
|
Definition
| Formed in OXIDIZING conditions; broken in REDUCING conditions (use reducing agent to break S-S) |
|
|
Term
|
Definition
Combination of SDS-PAGE and IEF; need to do IEF first, then SDS PAGE (otherwise SDS changes properties for IEF)
Determines the pI and MW of the unknown |
|
|
Term
| What wavelength do most normal proteins absorb at? |
|
Definition
| 280 nm; F, Y, W absorb here (increasing order) |
|
|
Term
|
Definition
| Amount of protein that catalyzes conversion of 1 micromole of substrate in 1 minute at 25 degrees Celsius |
|
|
Term
|
Definition
Enzyme units / total protein (mg)
Measure of purity |
|
|
Term
| Purification method by solubility... |
|
Definition
@ low salt - compensating charges cause electrostatic aggregation of proteins - proteins aggregate = LOW SOLUBILITY
@ increasing salt - salt coats the proteins to block compensating charges; increases solubility (stay in solution)
@ HIGH salt - salt ions coat protein and water molecules; less H2O can solvate the protein so hydrophobic effect becomes more pronounced @ high salt (proteins aggregate due to hydrophobic effect & precipitate = low solubility) |
|
|
Term
| How can pH be adjusted to alter solubility? |
|
Definition
pH < pI - protein is NET POSITIVE (protonated); proteins repel in solution and do not aggregate (high solubility)
pH = pI - protein is NET NEUTRAL; proteins aggregate due to hydrophobics and precipitate out (low solubility)
pH > pI - protein is NET NEGATIVE; like charges repel and remain in solution (high solubility) |
|
|
Term
| When is a protein most/least soluble in solution with its pI relative to the pH? |
|
Definition
If pH < pI - then protein is positive; high solubility
If pH > pI - then protein is negative; high solubility
If pH = pI - protein is neutral; low solubility as protein aggregates due to hydrophobic effect |
|
|
Term
| Anion Exchange vs. Cation Exchange |
|
Definition
Anion Exchange - have cations on column which BIND ANIONS
Cation Exchange - have anions on column which BIND CATIONS |
|
|
Term
| What charge exists on the column in cation exchange chromatography? |
|
Definition
| In cation exchange have a NEGATIVELY charged column |
|
|
Term
| What type of molecule is bound to the beads in CATION exchange? |
|
Definition
| Carboxyl methyl cellulose - acetyl group |
|
|
Term
| What occurs to proteins in a column when the salt concentration is increased? |
|
Definition
| At high salt concentrations, the charges on the column beads are shielded - this causes the protein to elute more quickly from the column |
|
|
Term
| What type of molecule is used in anion exchange? |
|
Definition
| (Et)3NH; have positive charge on nitrogen |
|
|
Term
| Hydrophobic Chromatography |
|
Definition
More hydrophobic proteins ELUTE SLOWER; typically use a benzyl group on the column (hydrophobic)
In this case, start with HIGH SALT (stronger hydrophobic effect), then slowly decrease salt concentration to elute proteins |
|
|
Term
| Different effects of increasing [salt] in ion exchange vs. hydrophobic chromatography |
|
Definition
Ion Exchange - at high salt, charges are shielded and the proteins elute more quickly
Hydrophobic - at high salt, the hydrophobic effect is more pronounced and the proteins take longer to elute |
|
|
Term
| Size Exclusion Chromotography |
|
Definition
| LARGEST proteins exit first (shortest path through porous beads); smaller proteins enter more pores = longer path - elute more slowly |
|
|
Term
|
Definition
| Volume at which the largest proteins elute from in size exclusion; this is the volume outside of the beads |
|
|
Term
|
Definition
| = volume within beads + volume outside of beads |
|
|
Term
| What is the ratio of elution volume/void volume dependent on? |
|
Definition
The protein's shape!
Rod shaped proteins behave like larger proteins in size exclusion chromatography because they can enter beads sideways as well |
|
|
Term
|
Definition
| Create solid phase which selectively binds to only one protein - attach specific LIGAND to beads which will be bound by the protein(s) in the mobile phase |
|
|
Term
| How to elute the column in affinity chromatography? |
|
Definition
| Flow in the same ligand that is attached to the beads in a "free form" - elute this free ligand through the column so that its binding competes with the binding of the ligand on the column |
|
|
Term
| What is the Edman reagent? |
|
Definition
| Phenylisothiocyanate (benzene-N=C=S) |
|
|
Term
| What does PITC react with? |
|
Definition
| All free amino groups (includes those at the N terminus, AND on the side chain of Lysine) |
|
|
Term
| What are the limitations of Edman degradation? |
|
Definition
PITC reacts twice with Lys side-chains; however only reaction with N-terminus produces the cyclization reaction and PTHaa derivative
Limit of less than 100 aa's
Often there is no free N-terminus (may be acetylated) |
|
|
Term
| Cleavage sites of the serine proteases? |
|
Definition
Trypsin - after K, R
Chymotrypsin - after F, Y, W
Elastase - after G, A, S, V
****HOWEVER, NONE cut before Pro |
|
|
Term
| Cleavage sites of non-serine proteases |
|
Definition
V8 Protease - after D, E
Asp-N - before D, E |
|
|
Term
| Method of non-enzymatic cleavage of a pp chain? |
|
Definition
| CNBr (acts on Met side-chain of following peptide bond; gives new free N-terminus) |
|
|
Term
| What are the problems with using amino acid composition determination by boiling the aa's in 6M HCl for 24 hrs? |
|
Definition
- All Trp is destroyed
- All amide bonds are broken (all peptide bonds and Asn/Gln is convered to Asp/Glu) |
|
|
Term
|
Definition
| Two proteins who show at least 25% sequence similarity; their similarity implies common ancestry |
|
|
Term
| Difference between PARALOGUES and ORTHOLOGUES (both are examples of HOMOLOGS) |
|
Definition
Paralogues - formed by DIVERGENT evolution; 2 similar proteins in 1 organism; 2 proteins usually have different cellular functions
Orthologues - 2 similar proteins in DIFFERENT organisms, but which share the same functions |
|
|
Term
| Example of DIVERGENT evolution |
|
Definition
| The globin gene - diverges into myoglobin, alpha & beta globin, gamma globin (in fetuses) |
|
|
Term
|
Definition
| See NON-SIMILAR PROTEINS (not homologs) in different organisms which share similar functions |
|
|
Term
| What are protein folds (tertiary structure) dependent on? |
|
Definition
| The primary structure of the pp chain (as shown by Anfinsen) |
|
|
Term
| True or false - proteins typically exist in many native conformations. |
|
Definition
FALSE
"Proteins typically exist in FEW native conformations" |
|
|
Term
|
Definition
| Spatial arrangement of atoms in the protein with respect to one another; conformation dictates where atoms are in relation to each other in 3D space |
|
|
Term
| What interactions contribute to protein folding? |
|
Definition
HYDROPHOBIC - predominates protein folding (entropic effect); important in tertiary structure development
Electrostatics - associations between compensating charges
H-bonding - very important for secondary structure |
|
|
Term
| What type of interaction is most important in determining tertiary structure? |
|
Definition
|
|
Term
| What type of interaction is most important in determining secondary structure? |
|
Definition
|
|
Term
| What type of conformation does the peptide bond possess? |
|
Definition
|
|
Term
| What effects does resonance have on the peptide bond? |
|
Definition
Due to resonance effects, the peptide bond has partial double bond character (partial negative dipole on O and partial positive dipole on N)
Because of this, there is NO rotation about the peptide bond |
|
|
Term
| For all aa's except for Pro, what is the conformation of the two side chains in relation to each other around the peptide bond? |
|
Definition
| For most amino acids, they are in TRANS conformation - this is most energetically favorable because it minimizes steric clashes between the two side-chains |
|
|
Term
|
Definition
PHI bond -> between N and alpha carbon
PSI bond -> between C of carbonyl group & alpha carbon
See free rotation about these two bonds/dihedral angles |
|
|
Term
| On what side of the amino acid are the two dihedral angles? |
|
Definition
|
|
Term
| What is the amount of conformationally accessible space as shown on a Ramachandran plot? |
|
Definition
| Only 25% of all dihedral angles are possible (only have 25% accessible space in conformations) |
|
|
Term
| Which bond is fixed in Pro? |
|
Definition
| The PHI bond (N-alpha carbon); it is fixed because the curving of the side-chain back around fixes it in place |
|
|
Term
| Relative amounts of a particular Pro residue in TRANS vs. CIS |
|
Definition
TRANS = 95-98% of the time
CIS = 2-5% of the time
NO INVERSION between trans & cis (stays as either one permanently) |
|
|
Term
| Why is there more Pro in cis than other amino acids? |
|
Definition
| Because when comparing the energy states for the trans and cis conformations of Pro, there is a very little difference; see a little difference in steric hindrance w/ Pro & other side-chains when looking at trans & cis |
|
|
Term
| Where is Pro often found? |
|
Definition
| At the end of helices (helix breaker) and in loops (B-turn) |
|
|
Term
|
Definition
Organized, LOCAL spatial arrangement of the polypeptide backbone; defined by often repeated PHI and PSI angles
Examples - alpha-helix, beta sheet, beta turn |
|
|
Term
| What is the simplest arrangement of amino acids in a pp chain with a full complement of H-bonds via backbone N's and O's? |
|
Definition
|
|
Term
| Properties of the a-helix |
|
Definition
Pitch = 5.4 Angstroms
3.6 residues/turn
R-handed helix |
|
|
Term
| Pattern of H-bonding in a-helix |
|
Definition
Every backbone C=O group H-bonds to a backbone NH group that is 4 residues down the helix (i to i+4 rule)
Remember that there are 3.6 residues/turn, so approximately 1 H-bond per turn in the helix |
|
|
Term
| Where are the H-bonds positioned relative to the helical axis? |
|
Definition
| H-bonds are PARALLEL to helical axis (backbone NH and C=O groups are also parallel to the axis) |
|
|
Term
| Which direction do the NH and C=O groups point, respectively? |
|
Definition
NH - all point UP towards the N-TERMINUS
C=O - all point DOWN towards the C-TERMINUS |
|
|
Term
| What is unique about the first 4 amino acids in the helix? |
|
Definition
| Can only act as H-bond ACCEPTORS (C=O pointing towards C-terminal) |
|
|
Term
| What is unique about the last 4 amino acids in the helix? |
|
Definition
| Can only act as H-bond DONORS (NH groups pointing up towards the N-terminus) |
|
|
Term
| What does the resonance of the peptide bond produce in the helix? |
|
Definition
Resonance creates partial dipoles on peptide bond - this creates electric dipoles on the helix too
N-terminus = POSITIVE
C-terminue = NEGATIVE
Overall helical dipole |
|
|
Term
| PHI and PSI angles for a-helices |
|
Definition
PHI = -60
PSI = -40
In the bottom skinny section on Ramachandran plot |
|
|
Term
| What is the stability in the a-helix due to in large part? |
|
Definition
| The regular H-bonding pattern throughout |
|
|
Term
| Relation of the R-groups to the helical axis? |
|
Definition
| R-groups point out perpendicularly to helical axis - decorate the helix |
|
|
Term
| What is the most frequently seen aa in helices? |
|
Definition
| Ala - due to small R group so it is easy to pack into the helical structure |
|
|
Term
| What is the least common aa in the helix and why? |
|
Definition
Proline
This is because of the R-group structure; sterically it is too big to incorporate into normal helical turns, also the PHI bond is fixed because of the R group
Also, Pro has no backbone amide proton (cannot be an H-bond donor, only acceptor) |
|
|
Term
| What other aa is also uncommon in helices? |
|
Definition
| Glycine - very flexible because of the R-group; can be difficult to lock into a single conformation in the helical structure |
|
|
Term
| How many degrees/amino acid residue int he helical wheel? |
|
Definition
3.6 residues per turn & 360 degrees/turn
= 100 degrees/residue |
|
|
Term
| What is the appearance of the protein backbone in beta structure? |
|
Definition
|
|
Term
| Difference in formation of secondary structure of B-structure and a-helices? |
|
Definition
a-helices can form on their own (via H-bonds); B-structure NEEDS certain regions within the pp sequence
2 sequences/structures needed in B-structure do NOT need to be in close proximity (in a-helices, all 1 continuous sequence so all is close) |
|
|
Term
| Arrangement of the 2 B-strands to form a sheet can be... |
|
Definition
| Parallel or anti-parallel |
|
|
Term
| What stabilizes the conformation of the B-sheet? |
|
Definition
| H-bonds (H-bonds are crucial to secondary structure) |
|
|
Term
| In the B-sheet what direction are the H-bonds relative to the backbone? |
|
Definition
H-bonds are PERPENDICULAR to the backbone
In contrast with a-helices which have H-bonds parallel to helical axis & backbone |
|
|
Term
| When considering stability arising only from H-bonds in B-sheets, which conformation is more stable? |
|
Definition
The ANTI-PARALLEL conformation is more stable because of the better alignment of H-bonds
In anti-parallel scenario, H-bonds are perfectly perpendicular to backbone; in parallel scenario, H-bonds are angled |
|
|
Term
| What is unique about loop regions compared to normal secondary structural elements? |
|
Definition
| Loop regions do not have defined & repeating PHI and PSI angles |
|
|
Term
| What is the shortest method to connect 2 antiparallel B-strands? |
|
Definition
| A B-turn (quick turn made up of 4 residues) |
|
|
Term
| What is the shortest method of connecting 2 parallel B-sheets? |
|
Definition
|
|
Term
| Difference between B-turn and random coils/loops? |
|
Definition
| The B-turn has defined PHI and PSI angles - it is a DEFINED secondary structural element (true secondary structure) |
|
|
Term
|
Definition
Need at least 1 H-bond between residues 1 and 4 of turn
4 residues make up the turn = shortest length that can turn 180 degrees in the backbone |
|
|
Term
| What are residues 2 and 3 often in the B-turn? |
|
Definition
Pro and Gly
Pro - because restricted PHI bond results in change in direction of chain
Gly - doesn't have a bulky side chain, so it can adopt a wider range of PHI and PSI angles (more flexible) |
|
|
Term
| What type of interactions maintain/stabilize a protein's conformation? |
|
Definition
Hydrophobics, electrostatics, H-bonding
S-S bonds (disulfides only function to maintain the fold, DO NOT assist in the folding process) |
|
|
Term
| What is true about the number of weak interactions and the free energy state of the protein? |
|
Definition
| Typically, the lowest free energy state is the one which MAXIMIZES the total number of weak interactions in the protein |
|
|
Term
| Which weak interactions generally predominate when considering maintaining protein stability? |
|
Definition
| Hydrophobics - see clustering of hydrophobic residues at the core of proteins to prevent contact with water (aggregation is entropic and due to hydrophobic effect) |
|
|
Term
| Where are salt bridges more stabilizing? |
|
Definition
| When they are in the interior of a protein (lower dielectric cosntant) |
|
|
Term
| How many covalent bonds separate adjacent amino acids? |
|
Definition
| 3 - the psi bond, the peptide bond, and the phi bond |
|
|
Term
| What is the simplest form of secondary structure the pp backbone can assume? |
|
Definition
|
|
Term
| R-groups in relation to helical axis of a-helices: |
|
Definition
| Protrude outwards (perpendicular to helical axis) - decorate the helix |
|
|
Term
| Where are positive and negative residues typically located in the helix in relation to each other? |
|
Definition
| Usually are 3-4 residues apart so that a salt-bridge (ion pair) can be formed by compensating charges |
|
|
Term
| Two reasons Pro is rarely seen in a-helices: |
|
Definition
- Phi bond is fixed due to side chain structure, so cannot assume proper phi angle for helix
- Backbone amide has no proton so it cannot participate in H-bonds |
|
|
Term
| Where are positive and negative amino acids typically found on an a-helix? |
|
Definition
Positive - located near C terminus
Negative - located near N terminus |
|
|
Term
| Factors affecting a-helix stability: |
|
Definition
Intrinsic property of primary structure to form helix (high degree of Ala, low Pro & Gly)
Interactions between R groups (e.g. ion pairs)
Interactions between residues @ end of helix and how this affects dipole moment of helix |
|
|
Term
| Differences beween H-bonds in a-helices and B-sheets? |
|
Definition
a-helices = H-bonds are parallel to the helical axis
B-sheets = H-bonds are located within the plane of the sheet, but form perpendicular to the individual sheets |
|
|
Term
| Are parallel or anti-parallel B-sheets typically more stable? |
|
Definition
| Anti-parallel because the bond angles and lengths for H-bonds formed are more ideal (remember H-bond directionality) |
|
|
Term
| Main type of structure seen in fibroins |
|
Definition
| Type of fibrous protein; mainly see B-structure; high content of Gly and Ala residues |
|
|
Term
| What type of sheets do B-turns link? |
|
Definition
| Link ANTI-PARALLEL sheets; shortest method to link 2 anti-parallel sheets |
|
|
Term
| Where do H-bonds form to stabilize the B-turn? |
|
Definition
| The C=O of the 1st residue with the HN of the 4th residue |
|
|
Term
| What two residues are commonly seen in B-turns and why? |
|
Definition
Gly - small & flexible
Pro - fixed phi bond and limited rotation allow for the turn to be made more easily
Seen often in residues 2 & 3 of the turn |
|
|
Term
| Difference between 2 and 3 structure? |
|
Definition
Secondary - spatial arrangement of residues in a LOCAL segment
Tertiary - looking at the LONG-RANGE aspects of the sequence which cause folding & have an associated function |
|
|
Term
| Characteristics of FIBROUS Proteins: |
|
Definition
Chain arranged into long strands/sheets; typically consist of only 1 type of secondary structure
Relatively simple tertiary structure
All are INSOLUBLE in water - high concentration of hydrophobic residues |
|
|
Term
| Usual functions of FIBROUS proteins |
|
Definition
| Protection, support, shape (a-keratin, collagen, fibroin) |
|
|
Term
| Characteristics of GLOBULAR proteins: |
|
Definition
Polypeptide chains FOLD BACK onto themselves to form a globular shape; more complex tertiary structure
Often shows multiple types of secondary structure
See a hydrophobic core and most are soluble in water |
|
|
Term
| What structures does a-keratin comprise? |
|
Definition
| Hair, wool, nails, claws, horns |
|
|
Term
|
Definition
Is a slightly distorted a-helix (see slight twisting in helical axis); there is also a slight compression in the helix
Pitch = 5.1 Angstroms; 3.5 residues/turn
The coil is slightly left-handed because of the curvature of the helical axis |
|
|
Term
| Relation of helix of a-keratin to normal a-helix |
|
Definition
Both are still R handed
Normal - 5.4 A, 3.6 residues/turn
a-Keratin - 5.1 A, 3.5 residues/turn
Note that the a-keratin helix is compressed due to the smaller pitch |
|
|
Term
| Structural Levels of a-Keratin |
|
Definition
Secondary - slightly compressed a-helix which is curved in a slight left-handed manner
Tertiary - dominated by a-helical secondary structure with the helical axis twisted slightly to the left (very hard to discern between 2 and 3 structure)
Quaternary - coiled coil that is made up of 2 supertwisted a-keratin helices with a slight L handed twist |
|
|
Term
| What governs the strength of a-Keratin (i.e. what holds adjacent coiled coils together)? |
|
Definition
| See covalent cross-links between coiled coils, stabilize quaternary structure (these links are S-S bonds) - more S-S bonds = stronger structure |
|
|
Term
| What does collagen make up? |
|
Definition
| CT - tendons, cartilage, matrix of bone |
|
|
Term
|
Definition
Known as an a-chain - left handed helix which is also slightly distorted by having a helical axis which is twisted slightly to the right
Pitch = 9.4 A, 3.3 residues/turn
Note: Helix is extremely stretched out to accomodate Pro and 4-Hyp into the structure |
|
|
Term
| Relative proportion of residues in collagen: |
|
Definition
| 35% Gly, 11% Ala, 21% Pro/4-Hyp |
|
|
Term
| What enzyme adds -OH to Pro to make 4-Hyp? |
|
Definition
| Prolyl 4-hydrolase - post translationally adds -OH group to form 4-Hyp |
|
|
Term
| Disease associated with collagen breakdown? |
|
Definition
Scurvy - due to inadequate vitamin C intake; prolyl 4-hydrolase requires vit C to function, so less intake means less 4-Hyp produced
This leads to breakdown of CT structures |
|
|
Term
| What type of interactions mainly stabilize globular proteins? |
|
Definition
|
|
Term
| Differences in structural levels between fibrous & globular proteins: |
|
Definition
2 - fibrous typically have 1 type of repeating secondary structure; globular usually have more than 1 type of secondary structure
3 - fibrous, usually quite simple and similar to 2 structure; globular, much more complex = more structural diversity |
|
|
Term
| Primary conclusion of the Anfinsen experiment? |
|
Definition
| That primary structure is the determinant for the folding of the protein (tertiary structure) |
|
|
Term
| How did Anfinsen show that primary structure dictates tertiary structure? |
|
Definition
By showing that the denaturation of proteins in vitro was reverisble (proteins could refold on their own)
Denatured ribonuclease A with urea & a reducing agent (B-ME) to remove S-S bonds, dialysis followed by removal of B-ME restored the protein's functional capability |
|
|
Term
| Hierarchical Model of Folding |
|
Definition
Folding goes from secondary to tertiary process step-wise
First local secondary structures form, followed by assembly of these structures into longer-range interactions between secondary structural elements (motifs), finally see domains and 3D folding occur |
|
|
Term
| Collapsed State Model of Folding |
|
Definition
Folding initiated by SPONTANEOUS collapse of the protein into a compact state (molten globule) - mediated by HYDROPHOBIC interactions
Many side chains of the globule remain dynamic and are not fixed |
|
|
Term
| Difference between motifs & tertiary structure? |
|
Definition
Motifs - are structural elements with NO associated function on their own
Tertiary Structure - has a given structure and designated function to go along with that structure |
|
|
Term
| Arrangement of secondary structural elements in B-a-B motif |
|
Definition
Two parallel B-strands (forming a B-sheet) with an a-helix above this parallel sheet
R groups from one face of helix, interact with R groups on one face of sheet (usually hydrophobically) |
|
|
Term
| Stability of the B-a-B motif |
|
Definition
H-bonds intrinsically stabilize the a-helix and parallel B-sheet
Also, get inter-structural interactions - interactions between the secondary structures are generally HYDROPHOBIC |
|
|
Term
| How many B-strands are in a B-barrel? |
|
Definition
| 8 parallel B strands connected by 7 a-helices |
|
|
Term
| Where are the a-helices located on the B-barrel? |
|
Definition
| On the outside of the barrel so they can interact w/ solvent (hydrophilic face would face the solvent) |
|
|
Term
| What joins the individual B-strands of the B-barrel? |
|
Definition
Strands 1&2-7&8 -> joined by a-helices connecting the parallel strands
Strands 1&8 -> joined by H-bonding between the two parallel strands |
|
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Term
| Structure/function relationship of B-barrel? |
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Definition
Formation brings amino acids important for function into close proximity (brings catalytic aa's close)
Center of B-barrel is the active site of the protein |
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Term
| What is the driving force for protein folding and stability? |
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Definition
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Term
| Why do S-S bonds form after protein folding? |
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Definition
| Formation requires strict angles and precise distances - formation does not occur randomly, so need the protein to be properly folded beforehand |
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Term
| Why are S-S bonds needed to maintain structure? |
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Definition
| Protection from the environment - when proteins are secreted from cells, the environment becomes much more harsh (maintain stability) |
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Term
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Definition
| Regions of primary structure where the polypeptide chain independently folds into a structural & functional unit |
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Term
| How are domains different from motifs? |
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Definition
| Domains have associated structure & function; on their own, motifs have NO function, need to be a part of a larger assembly |
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Term
| What is the benefit of having multiple domains in a given polypeptide chain? |
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Definition
Domains can functionally complement each other.
Do not need to create 3 separate chains and hope they eventually associate together - have all domains covalently linked to optimize function |
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Term
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Definition
| A protein that contains 2 or more polypeptide chains or subunits (for whole protein to be functional, need the individual subunits, aka protomers, to come together to form a complex) |
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Term
| Quaternary structure of Hb |
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Definition
| See two a-B heterodimers come together to form the final quaternary structure for Hb (2 a and 2 B subunits) |
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Term
| What are the typical shapes of FIBROUS proteins (how are pp's arranged)? |
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Definition
| Pp's are typically arranged in LONG STRANDS or in SHEETS (sheet-like structures; B-structure) |
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Term
| Solubility of fibrous vs. globular proteins in water |
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Definition
Fibrous - INSOLUBLE in water due to a high percentage of hydrophobic residues
Globular - SOLUBLE in water; hydrophobic residues are clustered at the core of the protein |
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Term
| Primary Structure of a-Keratin |
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Definition
See a HEPTAD repeat (a-b-c-d-e-f-g)
Residues a & d are usually HYDROPHOBIC and interact between the two a-helices in a coiled coil structure (hydrophobic interactions hold helices together in quaternary structure) |
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Term
| What governs the strength of an a-keratin fiber? |
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Definition
The extent of cross-linking between coiled coils in supramolecular structures -> cross-linking is covalently done by S-S bonds (more S-S bonds between coils = stronger structures)
See a higher percentage of Cys residues in horns (more Cys = more S-S bond formation) |
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Term
| Primary Structure of Collagen: |
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Definition
35% Gly, 21% Pro/4-Hyp, 11% Ala
See a tripeptide repeat - Gly-X-Y
X is usually Pro
Y is usually 4-Hyp |
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Term
| Why is the helix in collagen (a-chain) more extended compared to a normal a-helix? |
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Definition
It is more extended because the PHI bond of Pro/4-Hyp is fixed (side chain structure) so it cannot adopt the normal angle for an a-helix (normally phi = -60)
Therefore, this extends the pitch and causes more gradual turns (pitch of a-chain = 9.4 A) |
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Term
| Why can the 3 a-chains of the quaternary structure of collagen be packed very tightly? |
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Definition
| Because of the high percentage of Gly (35%) and Ala (11%) - small side chains so easily packed |
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Term
| Name of the supramolecular structures of collagen... |
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Definition
| FIBRILS (accumulation of supertwisted coiled coils |
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Term
| What links the 3 a-chains of the coiled coil of collagen? |
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Definition
| Non-covalent H-bonds that are perpendicular to the helical axis |
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Term
| Difference between H-bond stability in normal a-helix and a-chains of collagen |
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Definition
In a-helix, H-bonds stabilize secondary structure
In a-chains, H-bonds stabilize the quaternary structure of the coiled coil by holding chains together |
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Term
| What is essential for folding and maintenance of the triple helix coiled coil in collagen? |
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Definition
Post-translational modification to form 4-Hyp -> need addition of OH as it can also participate in H-bonding to stabilize the chains in the coil
Carbonyl group of Pro can also help H-bonding |
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Term
| What can be used to determine 3D structure of a protein? |
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Definition
NMR spec
X-ray crystallography |
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Term
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Definition
| The temperature/range of temperatures at which a protein is 50% unfolded (at 50% native structure) |
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Term
| What can we say about the folding/unfolding of a protein? |
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Definition
It is COOPERATIVE - cooperative interactions make up the folding process (interactions = non-covalent interactions)
See that disruption of some interactions causes cascade effect and disruption of many more (domino effect) |
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Term
| Why pH affects protein folding? |
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Definition
| Changes ionization states of the side chains - charged side chains will repel each other and destabilize the fold |
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Term
| Examples of chaotropic agents |
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Definition
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Term
| What property of chaotropic agents makes them particularly useful in protein unfolding? |
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Definition
They are highly soluble in water (lots of H bonding) -> can be taken to very high concentrations in water
By keeping them in high concentrations, the salts compete with protein moc for water; affects water surrounding the proteins to disrupt hydrophobic effect |
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Term
| What do chaotropic agents do to the protein in solution? |
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Definition
Denature (unfold it) - interact with water molecules surrounding protein to interfere with hydrophobic effect
Protein ends up unfolded, but still soluble in solution |
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Term
| What complicates the structure of ribonuclease A? |
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Definition
| The presence of 8 Cys residues giving rise to 4 S-S bonds |
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Term
| Process of denaturation of ribonuclease A in Anfinsen experiment: |
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Definition
1st to denature use 8M urea & B-ME as the reducing agent (unfold protein and cleave S-S bonds)
To renature, 1st do dialysis with UREA (need protein to refold first), THEN remove the reducing agent to allow S-S bond formation
Should see a complete return in function of the protein |
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Term
| What happens if B-ME is removed before the urea in the Anfinsen experiment? |
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Definition
Cys residues become oxidized BEFORE the protein folds; get a misfolded protein which may not be catalytically active
See formation of S-S bond oligomers |
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Term
| What does the collapsed state model emphasize? |
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Definition
| Protein dynamics - some secondary structure forms, but R groups are not fixed in position |
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Term
| What is the job of chaperone proteins in vivo? |
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Definition
| Provide a place for unfolded/misfolded proteins to fold or refold in an environment that PREVENTS unwanted aggregation or degradation |
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Term
| Diseases related to protein misfolding |
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Definition
Amyloidoses - Alzheimer's, Huntington's, DM type II
Bovine spongiform encephalopathy (misfolding of prion) |
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Term
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Definition
| Accumulation of amyloid fibrils in the brain; formed from a 40 aa fragment from a protein called the "amyloid precursor protein" (APP); fragment from APP = B-amyloid peptide |
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Term
| How is B-amyloid peptide formed? |
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Definition
It is proteolytically cleaved when APP is misfolded; cleaved at aa 42 in the sequence (1st 42 aa's become B-amyloid peptide)
Oligomeric forms may promote Alzheimer's |
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Term
| What is the infectious agent in BSE? |
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Definition
| Prion (proteinaceous infectious only) |
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Term
| How does misfolded prion lead to BSE? |
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Definition
| Misfolded prion (scrapie) interacts with normal prion and causes the normal prion to become unfolded -> see a domino effect where more and more native prion becomes unfolded to form large amyloid fibrils |
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Term
| What is the biochemical pathology in BSE? |
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Definition
| Misfolded prion forms amyloid fibrils (B-structure) which degenerate the brain |
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Term
| What does sequence conservation at a particular point in the protein imply? |
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Definition
| Implies that sequence likely has an important functional role for the protein |
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Term
| What type of quaternary structure is IgG said to have? |
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Definition
Is a heterotetramer (2 light chains and 2 heavy chains)
Light chains = 25 kDa; heavy = 50 kDa
Total weight = 150 kDa |
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Term
| How many Ig folds/domains are in 1 molecule of IgG? |
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Definition
| 12 domains (2 per light chain, 4 per heavy chain) |
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Term
| What is the Ig fold/domain structure? |
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Definition
B-sandwich -> have 2 antiparallel B-sheets stacked face to face
4 antiparallel B-strands on bottom sheet; 3 antiparallel B-strands on top sheet
Inside face of the sheets is HYDROPHOBIC (hydrophobic at center to prevent exposure to water)
S-S bond connects the sheets |
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Term
| What connects the 2 anti parallel B-sheets in the B-sandwich structure of the domains in IgG? |
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Definition
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Term
| How many hypervariable loops/moc of IgG? |
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Definition
| 12 (3 on each variable domain; 2 variable light chain domains, 2 variable heavy chain domains) |
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Term
| Where is the IgG most likely to be cut proteolytically? |
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Definition
| At the flexible (exposed) linker region (cut by proteases, e.g. papain) |
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Term
| What is released if the flexible linker region is cut? |
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Definition
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Term
| What is the structural function of the hypervariable loops in the variable domains of IgG? |
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Definition
| Connect the anti parallel B-strands in the variable domains of the light & heavy chains |
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Term
| How can we come up with different binding sites at the Fab regions? |
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Definition
| By varying the primary structure of the hypervariable loops (b/c primary structure governs tertiary structure) |
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Term
| What is the type of fit seen with the hypervariable loops at the antigen binding sites? |
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Definition
| INDUCED FIT - loops can slightly change strucute and are NOT part of regular secondary structure; often see a change in loop conformation when the antigen binds |
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Term
| Gene of variable region of IgG |
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Definition
300 V segments, 4 J segments 1 constant segment
The V and J elements are variable in different Ig's; get 1 V and 1 J per Ig |
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Term
| How many combinations are possible for the light chain of Ig's? Heavy chain? |
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Definition
Light = 3000 combinations
Heavy = 5000 combinations
Then multiply together, and account for mutations in the developing B cells to get 10^8 possibilities of different antibodies |
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Term
| Where does the variability in the Ig occur? |
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Definition
| In the Fab segments (antigen binding); Fc is constant for all Ig's |
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Term
| Where does the variability in Fab segments come from? |
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Definition
| Differences in primary structure of the hypervariable loops adjoining the B-strands of the variable regions |
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Term
| Which part of the polypeptide is ALWAYS involved in H-bonding? |
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Definition
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Term
| Relative bond length of peptide bond? |
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Definition
Intermediate (in between C-N and C=N bond lengths)
Implies that it has partial double bond character |
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Term
| How many atoms are in the same plane in the peptide bond? |
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Definition
| 6 - 2 alpha carbons, carbonyl group (C and O), amide group (H and N) |
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Term
| What is the main influence of phi and psi angles in a polypeptide? |
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Definition
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Term
| Main contribution to the stability of the a-helix? |
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Definition
| The regular H-bonding pattern throughout the helix |
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Term
| True or False - The a-helix has a hollow center |
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Definition
FALSE
The center of the helix is actually occupied be van der Waal's contacts of atoms in the pp chain |
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Term
| Relative positions of R groups in parallel vs. anti-parallel B-sheets |
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Definition
| In both scenarios, R groups show the same oscillating pattern, projecting above and below the plane of the sheet |
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Term
| What is common about edge strands for ALL types of B-sheets? |
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Definition
| Can form H-bonds with other parts of protein or water molecules |
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