Protein Structure & Function Flashcards
What structures of integral membrane proteins can pass through membranes?
Non-polar α-helices (can be single or multi pass)
Amphipathic secondary structures (membrane β-barrels)
Definition
a normal cell-surface glycoprotein that is conformationally characterized by two alpha helices and two complex-type N-linked oligosaccharide chains
PrPc
The bacterial flagellum…
Select one:
has the same evolutionary origin as Type IV pilus assembly
uses ATP hydrolysis as a source of energy for rotation
requires proton motive force for self-assembly but not for rotation
has a built-in Type II secretion system that exports rod, hook and filament components
uses Type III protein export for self-assembly
The bacterial flagellum…
Select one:
has the same evolutionary origin as Type IV pilus assembly
uses ATP hydrolysis as a source of energy for rotation
requires proton motive force for self-assembly but not for rotation
has a built-in Type II secretion system that exports rod, hook and filament components
uses Type III protein export for self-assembly
Define
Nanodiscs
a synthetic model membrane system which assists in the study of membrane proteins. It is composed of a lipid bilayer of phospholipids with the hydrophobic edge screened by two amphipathic proteins.
What are the disadvantages of protein therapeutics relative to small molecule therapeutics?
- Difficulty/cost of large-scale production
- Difficulty of purification
- Heterogeneity (inc. PTMs)
- Immunogenicity (if not natural human protein)
- Oral delivery not usually possible
- May degrade in plasma
- Reduced bioavailability: generally limited to extracellular targets
How has Insulin lispro and insulin aspart been altered?
They are mutated so that they tend not to self-associate, whcih facilitates more rapid absorption upon administeration
Definition
a large and complex molecular machine found primarily within the nucleus of eukaryotic cells
Spliceosome
Definition
misfolded proteins with the ability to transmit their misfolded shape onto normal variants of the same protein
Prions
Definition
a postulate in molecular biology that states that, at least for a small globular protein in its standard physiological environment, the native structure is determined only by the protein’s amino acid sequence
Thermodynamic hypothesis
What are the steps in protein structure determination by NMR spectroscopy?
- Express the protein; enrich in stable isotopes (15N, 13C)
- Optimise sample conditions (high conc, soluble)
- Assign the 1H, 15N and 13C signals in spectra
- Collect spectra to identify pairs of atoms that are close in space
- Use distance information to calculate a family of structures
- Iterate through previous steps to refine the structural ensemble
Define
Hit Rate
the number of active compounds per screen
Define
In silico Screening
performed on computer or via computer simulation
How do we know that effector proteins are unfolded when they pass through the T3SS needle?
The needle is a helical polymer of one protein with a diameter of 2nm, which is much smaller than most proteins so they must be unfolded when they thread through
Define
Spliceosome
a large and complex molecular machine found primarily within the nucleus of eukaryotic cells
What is QSAR used for?
Details of the role of various functional groups
Relative binding affinities of similar compounds
Models of pharmacophores for screening databases
X-ray crystallography is used for determining the three-dimensional structure of proteins by
Select one:
using radiofrequency pulses that perturb the magnetically susceptible nuclei.
using X-rays that are scattered by electrons.
using X-rays that are scattered by atomic nuclei.
using electrons that bounce off the surface of molecules in an ordered array.
using radiofrequency radiation to create diffraction patterns.
X-ray crystallography is used for determining the three-dimensional structure of proteins by
Select one:
using radiofrequency pulses that perturb the magnetically susceptible nuclei.
using X-rays that are scattered by electrons.
using X-rays that are scattered by atomic nuclei.
using electrons that bounce off the surface of molecules in an ordered array.
using radiofrequency radiation to create diffraction patterns.
Define
Amphipathic
(of a molecule, especially a protein) having both hydrophilic and hydrophobic parts.
Define
Bioavailability
the proportion of a drug or other substance which enters the circulation when introduced into the body and so is able to have an active effect
Define
Quantitative structure activity relationship (QSAR)
involves detailed computer modelling of the properties of a ligand or lead compound and the correlation of the information with activity data
Define
Van de Waals interactions
driven by induced electrical interactions between two or more atoms or molecules that are very close to each other
Define
Insulin glargine
a long-acting form of insulin used for the treatment of hyperglycemia caused by Type 1 and Type 2 Diabetes
How is SP cryo-EM performed?
- Freeze the sample (vitrobot)
- Place grid into a Transmission Electron Microscopy (TEM) that has a cryostage
- Collect data
Define
Pan-Assay Interference Compounds (PAINS)
chemical compounds that often give false positive results in high-throughput screens
What is the name of the sole member of the C-chemokine family?
Lymphotactin
A high B-factor indicates what?
High mobility
How do people think that prion gene mutations cause disease?
The presence of prions lowers the actiation energy of the conversion of the normal protein to the prion, thus greatly increasing the probability that this conversion will happen within the average lifespac
In the process of engineering insulin, several strategic amino acid changes have been made to produce fast-acting or long-acting insulin. Below is a schematic showing the amino acid sequence of the two chains of insulin. Highlighted are amino acids known to interact with the insulin receptor, occur at the dimer interface or have been modified in engineered forms of insulin.
Which of the following changes could lead to a faster acting insulin without affecting receptor binding?
Select one:
A21-Asn-> Lys
B16-Tyr -> Arg
B19-Cys -> Ala
B30-Thr -> Ser
B28-Pro -> Asp
In the process of engineering insulin, several strategic amino acid changes have been made to produce fast-acting or long-acting insulin. Below is a schematic showing the amino acid sequence of the two chains of insulin. Highlighted are amino acids known to interact with the insulin receptor, occur at the dimer interface or have been modified in engineered forms of insulin.
Which of the following changes could lead to a faster acting insulin without affecting receptor binding?
Select one:
A21-Asn-> Lys
B16-Tyr -> Arg
B19-Cys -> Ala
B30-Thr -> Ser
B28-Pro -> Asp
How has Insulin determir been altered?
It is acylated, resulting in reversible binding to albumin and slower absorption from tissue (distributed more slowly to tissue)
The lower the Kd, the ______ the affinity
The lower the Kd, the higher the affinity
Define
Glycosaminoglycans (GAGs)
long linear (unbranched) polysaccharides consisting of repeating disaccharide (double sugar) units
Which form of lympotactin binds to the receptor?
Monomer
Which of the following is NOT a step used in protein structure analysis by single particle cryo-electron microscopy?
Select one:
obtaining a monolayer of frozen protein particles
using an X-ray beam to obtain “shadow” images of the particles
superposition of many images of the particles detected at the same angle
collection of many images of single particles obtained at different angles
processing of images obtained at different angles to produce a three-dimensional model
Which of the following is NOT a step used in protein structure analysis by single particle cryo-electron microscopy?
Select one:
obtaining a monolayer of frozen protein particles
using an X-ray beam to obtain “shadow” images of the particles
superposition of many images of the particles detected at the same angle
collection of many images of single particles obtained at different angles
processing of images obtained at different angles to produce a three-dimensional model
Why are proteins frozen in different orientations for SP cryo-EM?
In order to develop a 3D image of the protein
Structure determination by NMR spectroscopy DOES NOT involve…
Select one:
radiofrequency pulses applied to a sample placed in a large magnetic field.
detection of radiofrequency signals derived from the electrons in the sample.
determination of which protons (1H) are close to which other protons in the sample.
calculation of three-dimensional structures that are consistent with determined distance constraints.
superimposition of three-dimensional structures calculated using the NMR data to check for consistency.
Structure determination by NMR spectroscopy DOES NOT involve…
Select one:
radiofrequency pulses applied to a sample placed in a large magnetic field.
detection of radiofrequency signals derived from the electrons in the sample.
determination of which protons (1H) are close to which other protons in the sample.
calculation of three-dimensional structures that are consistent with determined distance constraints.
superimposition of three-dimensional structures calculated using the NMR data to check for consistency.
What type of information does SAR provide?
Qualitative information
Define
Hydrogen bonds
the electromagnetic attraction created between a partially positively charged hydrogen atom attached to a highly electronegative atom and another nearby electronegative atom
What is used to determine the position of nuclei for the PDB?
Electron density detected through diffraction
Definition
a long-acting insulin analogue with a flat and predictable action profile
Insulin detemir
Define
Pharmacophores
a part of a molecular structure that is responsible for a particular biological or pharmacological interaction that it undergoes
_________ can be used to determine the number of binding sites and the affinity of each
Binding curves can be used to determine the number of binding sites and the affinity of each
Protein-ligand interactions…
Select one:
usually occur with a Kd in the picomolar range
usually occur through the formation of covalent bonds
are usually in equilibrium between bound and unbound forms
can reach 100% bound protein as long as enough ligand is added
occur through mainly hydrophobic interactions
Protein-ligand interactions…
Select one:
usually occur with a Kd in the picomolar range
usually occur through the formation of covalent bonds
are usually in equilibrium between bound and unbound forms
can reach 100% bound protein as long as enough ligand is added
occur through mainly hydrophobic interactions
Define
Insulin aspart
a rapid-acting form of insulin used for the treatment of hyperglycemia caused by Type 1 and Type 2 Diabetes
Why is NMR good for smaller molecules than for larger molecules?
The signal decays over time so in large molecules the signal is gone before it can be measured. The signal for smaller molecules lasts longer and therefore can be detected
What is the basic structure of T3SS?
What is the smallest unit of a lattice called?
Unit cell
Define
Immunogenicity
the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human and other animal
When two chemical fragments are identified that bind adjacently in a target active site, with Kd = 100 micromolar and 2 micromolar respectively (as illustrated below), their covalent attachment would be predicted to result in a compound…
Select one:
of reduced specificity
of Kd for the target equivalent to the sum total of the fragment affinities
of improved solubility
that binds to the target with greater than an additive affinity of the fragments
that is easier to synthesise than the separate fragments
When two chemical fragments are identified that bind adjacently in a target active site, with Kd = 100 micromolar and 2 micromolar respectively (as illustrated below), their covalent attachment would be predicted to result in a compound…
Select one:
of reduced specificity
of Kd for the target equivalent to the sum total of the fragment affinities
of improved solubility
that binds to the target with greater than an additive affinity of the fragments
that is easier to synthesise than the separate fragments
Which method should be used to determine the structure of DNA and RNA?
DNA and RNA are better visualised by NMR since they don’t crystalise very well
They are more “floppy” than proteins and they are charged
Define
X-ray Crystallography
the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions
Definition
complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu
Type III secretion system
Define
Phase problem
the problem of loss of information concerning the phase that can occur when making a physical measurement
Definition
the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human and other animal
Immunogenicity
Definition
a factor that can be applied to the X-ray scattering term for each atom (or for groups of atoms) that describes the degree to which the electron density is spread out; an indication of mobility of an atom
B-factor
What are the three key methods used to determine protein structure?
X-ray crystallography
NMR
Electron microscopy
Definition
a small cytokine belonging to the C chemokine family
Lymphotactin (Ltn)
What is the difference between PrPc and PrPSc?
PrPc = normal glycosylated cell-surface protein found in many tissue (particularly nerve cells). Protein is not essential
PrPSc = misfolded, prion form of PrPc
Why can’t full injectosomes or spliceosomes be characterised using NMR or X-ray crystallography?
They are much larger than these techniques can analyse
What comes first?
Hits or Leads
Hits
True or False
The phase angle cannot be measured directly
True
Which has better resolution?
X-ray or NMR
X-ray
Define
Docking
computational creation of a protein-ligand complex by simulation
What is the problem with in silico screening?
99% accurate scoring
In a library of 1 million compounds, 990 real actives found and 9900 false actives found
How has Insulin glargine been altered?
It has an increased isoelectric point which makes it less soluble at physiological pH which slows its absorption into tissue
Which of the following is NOT an advantage of protein therapeutics over small molecule therapeutics? Protein therapeutics…
Select one:
can have high specificity for their target.
can be immunogenic.
tend to have low toxicity.
can replace dysfunctional proteins.
can be readily engineered.
Which of the following is NOT an advantage of protein therapeutics over small molecule therapeutics? Protein therapeutics…
Select one:
can have high specificity for their target.
can be immunogenic.
tend to have low toxicity.
can replace dysfunctional proteins.
can be readily engineered.
True or False:
For X-ray crystallography to work, the structures must be arranged in a lattice
True
What is Docking?
Computational creation of a protein-ligand complex by simulation. Relies on a detailed structure of the protein and particularly its active site
Which of the following is FALSE? Single Particle cryo-Electron Microscopy can be used to determine the structure of…
Select one:
proteins or protein complexes that are membrane bound
large proteins or protein complexes to low resolution
many conformational states of a protein or protein complex
all sized proteins and peptides to low resolution
proteins or protein complexes that can not be crystallized
Which of the following is FALSE? Single Particle cryo-Electron Microscopy can be used to determine the structure of…
Select one:
proteins or protein complexes that are membrane bound
large proteins or protein complexes to low resolution
many conformational states of a protein or protein complex
all sized proteins and peptides to low resolution
proteins or protein complexes that can not be crystallized
Definition
chemical compounds that often give false positive results in high-throughput screens
Pan-Assay Interference Compounds (PAINS)
What prevent polymerisation of flagellin molecules in the cytoplasm?
Chaperones
Which of the following chemical screening strategies will NOT increase the hit-rate in drug discovery?
Select one:
Avoiding compounds that have properties according to Lipinski’s rule of five.
Avoiding “Pan-Assay Interference” compounds that often appear as hits in assays.
The use of in silico screening of compounds to consider potential interactions with the target.
De novo design of chemical structures that have potential to fit in the active site of the target structure.
Fragment screening to identify chemical fragments that bind to the target as determined experimentally.
Which of the following chemical screening strategies will NOT increase the hit-rate in drug discovery?
Select one:
Avoiding compounds that have properties according to Lipinski’s rule of five.
Avoiding “Pan-Assay Interference” compounds that often appear as hits in assays.
The use of in silico screening of compounds to consider potential interactions with the target.
De novo design of chemical structures that have potential to fit in the active site of the target structure.
Fragment screening to identify chemical fragments that bind to the target as determined experimentally.
Define
PrPsc
an alternatively folded variant of the cellular prion protein, PrPC, which is a regular, GPI-anchored protein that is present on the cell surface of neurons and other cell types
Definition
involves detailed computer modelling of the properties of a ligand or lead compound and the correlation of the information with activity data
Quantitative structure activity relationship (QSAR)
Definition
the stationary part of a rotary system, found in electric generators, electric motors, sirens, mud motors or biological rotors
Stator ring
The B-factor is only meaningful in structures determined how?
Using X-ray diffraction
Why is one considered strong while the other is weak?
The electron orbitals of the oxygen overlap with the hydrogen for the strong one
Definition
driven by induced electrical interactions between two or more atoms or molecules that are very close to each other
Van de Waals interactions
How does the absorption rate of insulin change as the number of subunits (i.e. monomer, dimer) increases?
Monomor absorbs fastest
Monomer > Dimer > Tetramer > Zn-Hexamer
For most proteins, alternative native conformations exist in dynamic equilibrium, what is the exception?
Prions
Define
Nuclear magnetic resonance (NMR)
a physical observation in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field and therefore not involving electromagnetic waves) and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus
True or False:
Protein structures are fixed
False
Protein structures are dynamic
Definition
a protein structure found at the base of a eukaryotic undulipodium (cilium or flagellum)
Basal body
What does QSAR involve?
Detailed computer modelling of the properties of a ligand or lead compound and the correlation of this information with activity data (no knowledge of target required)
What type of amino acids does a multi pass α-helix have?
Amphipathic
i.e. having both hydrophillic and hydrophobic regions
The NMR signal is derived from what?
The radiofrequency signals of magnetically susceptible nuclei
Define
Prions
misfolded proteins with the ability to transmit their misfolded shape onto normal variants of the same protein
What are the problems with drug design?
- Biological systems are notoriously finicky
- Protein movements
- Adaptation to ligand
- Weak bonds very important
- One H bond = ~10x ligand affinity
- Non-standard bonds
- CH H bond donors, CF ‘fluorine’ bonds
- Protein movements
- Little mistakes in the structures = big mistakes in the models
Why is the (O=)C-N bond considered partially double?
Resonance. This prevents rotation around this bond making it planar
Which protein structure level does PrPc and PrPSc differ?
Secondary
Tertiary
(Quaternary)
What is the mathematical relationship between the fraction of P bound and the free concentration of L?
Fraction P bound = [L]/(Kd + [L])
Rearrange the dissociation constant equation
What is the proton motive force used for in the flagellum?
Energises the translocation of unfolded proteins through the channel in the flagellum during its self-assembly
Produces the ernergy for the rotation of flagellum
Define
Attrition
when participants leave during a study
The low resolution SP-cryo EM structure of the perforin pore shows that it is large enough for granzyme B to be…
Select one:
passively translocated in an unfolded state
actively translocated in an unfolded state
passively translocated in a folded state
actively translocated in a folded state
blocked from passing through the pore
The low resolution SP-cryo EM structure of the perforin pore shows that it is large enough for granzyme B to be…
Select one:
passively translocated in an unfolded state
actively translocated in an unfolded state
passively translocated in a folded state
actively translocated in a folded state
blocked from passing through the pore
What are the fast-acting insulin analogues?
Insulin lispro
Insulin aspart
Definition
the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions
X-ray Crystallography
Definition
antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans
Humanised therapeutic antibodies
What are the steps in protein structure determination by X-ray crystallography?
- Express/purify the protein
- Crystallise the protein (can’t be dry)
- Collect diffraction data
- Determine the space group, unit cell dimensions, and number/symmetry of molecules per unit cell
- Solve the “phase problem” (phase angle)
- Calculate an electron density map
- Build a molecular model to fit the electron density map
- Refine the model
Define
Basal body
a protein structure found at the base of a eukaryotic undulipodium (cilium or flagellum)
Definition
long linear (unbranched) polysaccharides consisting of repeating disaccharide (double sugar) units
Glycosaminoglycans (GAGs)
Why does the species barrier exist for prions?
The species barrier exists because a certain degrees of sequence identity/similarity between the host PrPc and the acquired PrPSc is required for the interaction and catalysis to occur
What techniques can we use if we have known ligands but no known target structure?
QSAR
Scaffold hopping
The basal body of the bacterial flagellum and bacterial injectisome is used to secrete monomers that, once outside of the cell, polymerise into the filament and needle, respectively. What prevents them from polymerisation inside the cell?
Select one:
Protein chain is fed into the secretion channel as it is being synthesized
They need metal ion for polymerisation, which is only available outside
They are held in an unfolded state by intracellular chaperones
They require energy of ATP hydrolysis to polymerise
They require proton-motive force to polymerise
The basal body of the bacterial flagellum and bacterial injectisome is used to secrete monomers that, once outside of the cell, polymerise into the filament and needle, respectively. What prevents them from polymerisation inside the cell?
Select one:
Protein chain is fed into the secretion channel as it is being synthesized
They need metal ion for polymerisation, which is only available outside
They are held in an unfolded state by intracellular chaperones
They require energy of ATP hydrolysis to polymerise
They require proton-motive force to polymerise
Definition
tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules
Hydrophobic interactions
Why is hydrogen atoms not displayed in X-ray crystallography?
Hydrogens can’t be seen because 1 electrion is often not enough to produce a signal
Which method currently contributes the most to the PDB database?
X-ray crystallography
Which of the two ligands has a higher affinity to protein?
Red
Below is a two-dimensional 1H NMR NOESY spectrum acquired for a protein. What do the crosspeaks (the signals that are not on the diagonal of the spectrum) represent?
These are signals that indicate that…
Select one:
a particular 1H is bonded to another particular 1H.
a ligand is bound to the protein.
one 1H is in two different electronic environments.
a particular 1H is close to other particular 1H through space.
the sample is also labelled with 15N.
Below is a two-dimensional 1H NMR NOESY spectrum acquired for a protein. What do the crosspeaks (the signals that are not on the diagonal of the spectrum) represent?These are signals that indicate that…
Select one:
a particular 1H is bonded to another particular 1H.
a ligand is bound to the protein.
one 1H is in two different electronic environments.
a particular 1H is close to other particular 1H through space.
the sample is also labelled with 15N.
Consider the small ligand molecule bound in a pocket in a protein shown below, and identify the binding interactions that are labelled i-iv.
The interaction labelled iv is:
Select one:
a salt bridge
a covalent bond
a hydrogen bond
a van der Waals contact
hydrophobic
Consider the small ligand molecule bound in a pocket in a protein shown below, and identify the binding interactions that are labelled i-iv.
The interaction labelled iv is:
Select one:
a salt bridge
a covalent bond
a hydrogen bond
a van der Waals contact
hydrophobic
Below is a 1H-15N HSQC spectrum acquired for an 15N-labelled protein. There is one signal for every NH group in the protein.
What do the contoured spots represent?
Select one:
signals from each and every 1H nucleus in the protein
signals from 1H and 15N nuclei that are covalently bonded to each other
signals from 1H and 15N nuclei that are close to each other through space
signals that arise due to ligand binding
What do the contoured spots represent?
Select one:
signals from each and every 1H nucleus in the protein
signals from 1H and 15N nuclei that are covalently bonded to each other
signals from 1H and 15N nuclei that are close to each other through space
signals that arise due to ligand binding
Below is the amino acid sequence of the region of an integral membrane protein that spans the membrane. Have a close look at the sequence and identify the amino acids that have non-polar side chains.
634EGCPTNGPKQPSIAVGIVGALLLLLVVALGIGLFIRRRHTNRKR677
Note the 20 amino acid structures shown below.
What kind of secondary structure is used to span the membrane?
Select one:
amphipathic β-strand
amphipathic α-helix
hydrophilic β-strand
nonpolar α-helix
What kind of secondary structure is used to span the membrane?
Select one:
amphipathic β-strand
amphipathic α-helix
hydrophilic β-strand
nonpolar α-helix
Below is the amino acid sequence of the region of an integral membrane protein that spans the membrane. Have a close look at the sequence and identify the amino acids that have non-polar side chains.
634EGCPTNGPKQPSIAVGIVGALLLLLVVALGIGLFIRRRHTNRKR677
Note the 20 amino acid structures shown below.
How many times does this protein sequence span the membrane?
Select one:
once
twice
three times
four times
How many times does this protein sequence span the membrane?
Select one:
once
twice
three times
four times
Below is the amino acid sequence of the region of an integral membrane protein that spans the membrane. Have a close look at the sequence and identify the amino acids that have non-polar side chains.
634EGCPTNGPKQPSIAVGIVGALLLLLVVALGIGLFIRRRHTNRKR677
Note the 20 amino acid structures shown below.
Look at the amino acid residue F667 and consider what would happen if it was substituted with a different residue. Which amino acid residue at this position is likely to be the least disruptive to the structure of this protein?
Select one:
aspartic acid
arginine
lysine
leucine
Look at the amino acid residue F667 and consider what would happen if it was substituted with a different residue. Which amino acid residue at this position is likely to be the least disruptive to the structure of this protein?
Select one:
aspartic acid
arginine
lysine
leucine
