Notes Flashcards
(54 cards)
When do you expect to form an alpha helix? Disordered secondary structure?
Alpha helix: The alpha helix is stabilized by intrachain hydrogen bonds between the carbonyl and amino groups of peptide bonds FOUR residues away.
The alpha helix is prevented from forming when two or more consecutive residues with like charges (eg. lysine, glutamate) or by two or more consecutive residues with bulky R groups that branch at the beta carbon (eg. isoleucine, threonine, valine), in these cases the polypeptide chain may assume a random coil structure.
Proline cannot participate in forming a alpha helix because the nitrogen atom is in a rigid ring.
Successive serine residues disrupt alpha helices because of tendency of the OH groups to hydrogen bond strongly to water.
What type of residues tend to form beta sheets?
Repeating sequences of amino acids with small, compact R groups (eg. glycine, alanine) tend to form beta sheets, structures which consists of parallel or antiparallel polypeptide chains linked by interchain hydrogen bonds.
Silk is antiparallel.
Calculate the axial length of an alpha helix containing 78 amino acids
How long would the polypeptide chain be if it were fully extended?
The alpha helix rises 5.4 A for every 3.6 residues (1.5 A per residue)
So 1.5 A x 78 = 117 A
In the fully extended chain, the distance between residues is 3.6 A, so
3.6 A x 78 = 280.8
Glycine is a highly conserved amino acid in the evolution of proteins. Why? What other amino acids might be expected to have similar evolutionary conservation?
Glycine have very unhindered dihedral angles (R group is very small).
Proline is also very conserved for the opposite reason. It’s dihedrals are particularly hindered because of a ring with the nitrogen atom.
Given only a primary structure, how predictable would H bonding patterns be for the different types of secondary structuer commonly observed in globular proteins?
Most predictable - Alpha helix - Antiparallel beta sheet - Parallel beta sheet - Disordered Not predictable
Why do you absolutely not want to lose the double bond character in a peptide bond between amino and carbonyl?
The double bond character is conferred through resonance. This is highly stabilizing and present in both cis and trans peptide bonds, though trans peptide bonds are more stable in general because of steric hindrance.
The leucine zipper is formed by the leucine sidechains repeated every 7th residue. It was hypothesized that this was by two alpha helices interacting through their sidechains intercalating. This is incorrect. What is the correct structure?
If the sidechains were interacting with each other then they would be interacting every 7 residues, but in alpha helices there would be sidechains every 7.2 residues (because there are 3.6 residues per turn). So distortion would occur to accommodate. This doesn’t happen, instead you observe a coiled coil structure, which is not alpha helical.
List the physical phenomena underlying light absorption in the various regions of the electromagnetic spectrum
X-rays: Subvalence electrons excited to higher energy levels
UV/Vis: Valence electrons excited to higher energy levels
IR: Molecular vibration
Microwave: Molecular rotation
In spectroscopy, the absorbance of a solution is a linear function of concentration. Doubling c results in a doubling of the absorbance, tripling c results in a tripling of the absorbance and so on. What happens to the transmission (I) when:
The concentration is doubled?
The path length is increased n-fold?
Concentration doubled: 2c: I^2
Path length increased n fold: nl: I^n
At what wavelengths to proteins absorb light?
Most proteins have a distinct absorption maximum at 280 nm due to tyrosine, tryptophan and phenylalanine. However, all proteins absorb strongly below 230 nm due to the peptide bond.
What is fluorometry?
Emitted fluorescence light is observed at 90 degrees to the incident light and two wavelength selectors are required (one to transmit the desired excitation wavelength and one to select the desired emission wavelength).
Fluorometry can be extremely selective since only certain wavelengths of light will excite a given compound.
The fluoresence emitted by one substance may be absorbed or quenched by other substances in the sample. For this reason it is necessary to include an internal recover standard in each assay.
Define the Boltzmann distribution
In physics and mathematics, the Boltzmann distribution is a certain distribution function or probability measure for the distribution of the states of a system.
Consider the following primary sequence
Ala-Ile-Arg–Ser-Phe-Ala-Glu
Protein A: Alpha helix
Protein B: Beta strand
Would you expect to see significant differences in a selective (Ile and Phe specific) TOCSY spectra for protein A vs. B?
TOCSY relies on J coupling and you observe connections between J coupled protons (protons in J coupled networks). Ile and Phe will yield fairly similar TOCSY spectra.
Because there is a quaternary carbon on Phe, there is only one CH2 and the amino that TOCSY will pick up.
What is J coupling?
Scalar or J-couplings (also called indirect dipole dipole coupling) are mediated through chemical bonds connecting two spins. It is an indirect interaction between two nuclear spins which arises from hyperfine interactions between the nuclei and local electrons.[1] J-coupling contains information about bond distance and angles. Most importantly, J-coupling provides information on the connectivity of molecules. In NMR spectroscopy, it is responsible for the appearance of many signals in the NMR spectra of fairly simple molecules.
Consider the following primary sequence
Ala-Ile-Arg–Ser-Phe-Ala-Glu
Protein A: Alpha helix
Protein B: Beta strand
How could you use NOESY experiments to distinguish protein A from protein B?
NOESY strictly depends on the distance between H bonding dipole-dipole. The cutoff for detection is 6 Angstroms.
Alpha helix will give numerous H-H NOE connections due to proximity in space (positive results). But The distance between Ile and Phe in beta strand is longer than 6 Angstroms due to R group positioning (negative results).
Consider an alpha helix, based upon your knowledge of the hydrogen bonding configuration and structure of an alpha helix, is there a patter and directionality of amide proton NOE contacts that you would predict?
The N-H would point N terminally in an alpha helix. That protein should be observing contacts that are N terminal.
NOE should be observed to residues N terminal to it and within 4 residues.
Engineer three proteins to make them easily distinguishable from each other by either optical or NMR spectroscopy.
- No aromatics (just peptide bond absorption)
- Phe-heavy (distinguishable but worse fluorescence)
- Trp Heavy (absorption and fluorescence)
You can use some NMR distinguishable amino acids as well (eg. some with methyl groups, some without etc. )
When is an enzyme catalyzed reaction in steady state?
When [S] > [E] and [ES] is formed at the same rate it decomposes and the reaction is at Vmax!
Michaelis-Menten kinetics ONLY apply to steady state reactions.
Give a very simplified description of Km
It is the rate of ES breakdown/rate of ES formation
It describes the dependence of Vo on [S] and the steepness of the curve on the Michaelis-Menten graph
Describe the importance of binding energy from enzyme-substrate interaction
The binding energy is the free energy released in the formation of a large number of weak interactions between the enzyme and the substrate. We can envision this binding energy as serving two purposes: it establishes substrate specificity and increases catalytic efficiency. Only the correct substrate can participate in most or all of the interactions with the enzyme and thus maximize binding energy, accounting for the exquisite substrate specificity exhibited by many enzymes. Furthermore, the full complement of such interactions is formed only when the substrate is in the transition state. Thus, interactions between the enzyme and the substrate not only favor substrate binding but stabilize the transition state, thereby lowering the activation energy. The binding energy can also promote structural changes in both the enzyme and the substrate that facilitate catalysis, a process referred to as induced fit.
List four catalytic strategies commonly employed by enzymes
1.
Covalent catalysis. In covalent catalysis, the active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism
2.
General acid-base catalysis. In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine (E-BH or E-B:)
3.
Metal ion catalysis. Metal ions can function catalytically in several ways. For instance, a metal ion may serve as an electrophilic catalyst (eg. Zn), stabilizing a negative charge on a reaction intermediate. Alternatively, the metal ion may generate a nucleophile by increasing the acidity of a nearby molecule, such as water in the hydration of CO2 by carbonic anhydrase. Finally, the metal ion may bind to substrate, increasing the number of interactions with the enzyme and thus the binding energy. This strategy is used by NMP kinases
4.
Proximity effect: Many reactions include two distinct substrates. In such cases, the reaction rate may be considerably enhanced by bringing the two substrates together along a single binding surface on an enzyme. NMP kinases bring two nucleotides together to facilitate the transfer of a phosphoryl group from one nucleotide to the other
What is a suicide inhibitor?
In biochemistry, suicide inhibition, also known as suicide inactivation or mechanism-based inhibition, is a form of irreversible enzyme inhibition that occurs when an enzyme binds a substrate analogue and forms an irreversible complex with it through a covalent bond during the “normal” catalysis reaction.
This usually uses a prosthetic group or a coenzyme, forming electrophilic alpha and beta unsaturated carbonyl compounds and imines.
Aspirin is a suicide inhibitor of COX1/COX2
What are three types of reactions that PLP can participate in? How do these reactions usually start?
Transamination
Decarboxylation
Racemization
All of these normally begin with the aldehyde group of the coenzyme forming an imine (Schiff base) linkage with a lysine side chain on the enzyme.
Recall the mechanism for disulfide formation
look it up
