Solutions to BIEN310 Assignments 1 & 2

A1, Q1 (1 points). What is the difference between forward engineering and reverse engineering? Can you give an
example in biomolecular engineering for each?

Forward engineering approaches aims to design biomolecular systems with novel functionalities. Examples of forward
engineering in biomolecular engineering are protein design and engineering, where new proteins with new
functionalities are designed and engineered, or synthetic biology, where new biomolecular circuits are designed and
engineered.
Reverse engineering approaches aims to understand naturally occurring complex biomolecular systems. Examples of
reverse engineering in biomolecular engineering are systems biology, where the “wiring diagrams” of the cell are
measured and used to understand how the cell functions, or structural biology, where the atomic positions and
interactions of the protein are measured and used to understand how the protein functions.

A1, Q2 (1 points). Go to the GenBank which is a database of genetic sequence information

(http://www.ncbi.nlm.nih.gov/genbank). Select the “GenBank” tab and then click “Statistics” to find details regarding
GenBank statistics. How many sequence records are in GenBank, excluding WGS (Whole Genome Shotgun)? How many
sequence records are from WGS?

Sequence records (GenBank): 201,663,568

Sequence records (WGS): 487,891,767

A1, Q3 (1 points). You are engineering new proteins for energy applications by tinkering with naturally occurring
proteins. You are specifically interested in getting data about cytochrome B in sperm whale (Physeter catodon), a
mitochondrial protein involved in the electron transport chain. Search for the DNA nucleotide sequence of sperm whale
cytochrome b (in GenBank) and open the GenBank search result. What is the value of ACCESSION field of the top-ranked
GenBank file? (NOTE: We are looking for the entry with complete cds available. “cds”, meaning “coding sequence”, is
part of a standard annotation vocabulary for DNA sequences. In this entry there are 1140 bases that code for protein.)

X75589 or AF304073

A1, Q4 (1 points). Search for the protein sequence of the sperm whale cytochrome B (in GenBank). What are the first
ten residues of the protein sequence associated with the top-ranked GenBank file?

mtnirkshpl

A1, Q5 (1 points). Other organisms might also have cytochromes with similar sequences to those in sperm whales.
BLAST is a tool that allows you to search for similar sequences among NCBI’s various sequence databases. Read about
BLAST at website: http://blast.ncbi.nlm.nih.gov/. Which one of the four BLAST methods do you want to use to search for
a protein sequence against a protein database (nucleotide BLAST, protein BLAST, tblastn, or blastx)?

Protein BLAST

A1, Q6 (1 points). Do a BLAST search against SwissProt database using the first 60 residues of the sperm whale
cytochrome B protein sequence. (NOTE: The results give a long list of “hits” along with scores. The cytochrome B
sequence has a large number of hits in many different organisms indicating that the cytochrome B sequence is very
conserved.) The first hit is the sperm whale protein sequence from which the query was generated. Look at the second
best alignment. What organism is the second best alignment from?

Ziphius cavirostris (Cuvier's beaked whale)

A1, Q7 (1 points). Go to the Protein Data Bank (PDB), which is a database of macromolecular structural information
(http://www.rcsb.org). Search the Protein Data Bank for the record 3GB1, which is the structure for a naturally-occurring
protein. PDB offers several visualization programs to look at the protein structure. We will be using JSmol. Click “View in
3D: NGL or JSmol (in Browser)” beneath the picture of the protein structure, and JSmol should launch automatically. The
protein structure should automatically be in cartoon view. (If not, right click on the image and go to Style -> Structures ->
Cartoon menu, then left click.) Dragging your mouse over the image rotates the structure. How many alpha-helices are
in this protein structure? How many beta-strands? Do beta-strands form parallel beta-sheets, anti-parallel beta-sheets,
or mixed?

There are 1 alpha-helix and 4 beta-strands. Beta-strands form mixed beta-sheets.

A1, Q8 (1 points). Search the Protein Data Bank for the record 1QYS, a computationally designed protein with a
completely novel structure never seen in nature. How many alpha-helices are in this protein structure? How many beta-
strands? Do beta strands form parallel beta-sheets, anti-parallel beta-sheets, or mixed?

There are 2 alpha-helix and 5 beta-strands. Beta-strands form antiparallel beta-sheets.

A1, Q9 (2 points). Search the Protein Data Bank for the record 1ZRC, which is the 3D structure for the E. coli CAP protein
(catabolite activator protein) bound to DNA. What experimental method was used to obtain the protein structure? The
CAP protein contains both alpha-helices and beta-sheets. There should be an obvious secondary structure visibly
interacting with the DNA major groove. What is it?

X-ray diffraction/crystallography. Alpha-helix.

A2, Q1 (1 points). Select all statements below which are true for protein secondary structure:
(A) Secondary structure is stabilized by hydrogen bonding of backbone atoms.
(B) Secondary structure is stabilized by hydrogen bonding of side chain atoms, but not backbone atoms.
(C) Secondary structure conformations minimize steric clashes on the Ramachandran diagram.
(D) Secondary structure contents do not vary much among different proteins.

(A), (C)

A2, Q2 (1 points). Select all statements below which are true:

(A) Hydrophobic interaction is a main driving force of folding for water-soluble proteins.
(B) Hydrophobic interaction is a main driving force of folding for transmembrane proteins.
(C) Protein interiors are as close-packed as liquid water.
(D) The main driving force of protein folding for water-soluble proteins is not explicitly included as a term in the
molecular dynamics “force field”.

(A), (D)

A2, Q3 (2 points). You are designing a transmembrane ion channel protein that will only allow chloride ions (Cl-) to pass
through the membrane. A key task is to carefully arrange the backbone atoms of the protein so as to stabilize the
chloride ion in the central cavity of the protein. This way, the protein machine scrutinize the ion to make sure that it is
indeed chloride before passing it through the membrane. (A) Which secondary structure would you use to accomplish
this goal (alpha helix, beta strand/sheet, or loop/turn)? Briefly explain why. (B) Would you point the N-terminal or the C-
terminal of the said secondary structure towards the chloride ion? Briefly explain why.

(A) Alpha helix, as it carries a large dipole moment. (B) I would point the N-terminal of the alpha helix towards Cl- ion,
as N-terminal is positively charged.

A2, Q4 (1 points). The electrostatic interaction between two point charges is weakened in the presence of a dielectric
material in the following way: U = (4πε0κ)-1q1q2/r, where the dielectric constant κ is 1 for vacuum, 20 for protein, and 80
for water. You are carrying out an all-atom molecular dynamics simulation of a 1,000-atom protein in a box of 1,000
water molecules. What dielectric constant value(s) should you use for the electrostatic interaction term in the molecular
dynamics “force field”? Briefly explain why.

κ=1 for vacuum should be used. Vacuum electrostatic interaction is a fundamental interaction explicitly included in
the molecular dynamics “force field”. In contrast, dielectric effect (of protein & water) is a complex interaction not
included in the “force field”, but it spontaneously emerges out of the molecular dynamics simulation.

A2, Q5 (1 points). Calculate the ratio of the electrostatic attraction force (in vacuum) to the gravitational attraction force
between a pair of potassium (K+) and chloride (Cl-) ions separated at a distance r. Which force is larger, and by how many
times? According to your calculation, can gravitational forces be safely ignored in molecular dynamics simulations?
Recall that Coulomb’s constant is ke = (4πε0)-1 = 9.0x109 Nm2C-2, gravitational constant is G = 6.7x10-11 m3kg-1s-2, the
magnitude of one electron charge is 1.6x10-19 C, the atomic masses of K+ and Cl- are 39 and 35 a.m.u. respectively, where
1 a.m.u. = 1.7x10-27 kg.

fe/fG = (keq1q2)/(Gm1m2) = (9.0x109 Nm2C-2 x 1.6x10-19 C x 1.6x10-19 C) / (6.7x10-11 m3kg-1s-2 x 39x1.7x10-27 kg x

35x1.7x10-27kg) = 8.7x1032. Electrostatic force is much larger, and gravitational force can be ignored.

A2, Q6 (2 points). You are designing a new protein with many strong and weak interactions, and you want to know if
strong or weak interactions store more energy. To investigate this problem, you consider a “toy” protein with just one
atom with mass m (see figure below). The atom can only move along the x direction. The atom is connected to a strong
spring A to the left, and a weak spring B to the right. Thus kA >> kB, where kA and kB are the spring constants for A and B,
respectively. An atomic force microscope grabs the free ends of the two springs and stretches them before stopping. A
mechanical equilibrium is reached so that the atom no longer moves. At this mechanical equilibrium, the two springs are
stretched with displacements of xA and xB, respectively. The potential energies stored in the two springs are UA and UB,
respectively. (A) Calculate the total potential energy of the whole protein system Ut, and write it in terms of kA, kB, xA, xB,
and m. (B) Calculate the ratio of xA to xB, and write it in terms of kA, kB, and m. (C) Calculate the ratio of UA to UB, and
write it in terms of kA, kB, and m. (D) Do strong or weak interactions store more energy in proteins? Briefly explain why.

(A) Ut = kAxA2/2 + kBxB2/2

(B) kAxA = kBxB, so xA/xB = kB/kA
(C) UA/UB = kB/kA
(D) Weak interactions store more energy

A2, Q7 (2 points). Pure membranes of dipalmitoyl lecithin phospholipids are models of biological membranes. They melt
at the temperature of 41 oC, and the amount of heat absorbed during melting is 9 kcal mol-1. (A) Calculate the entropy
increase of melting, ΔS. (B) There are 32 rotatable CH2-CH2 bonds in each molecule. What is the increase in multiplicity
on melting one mole of bonds?

(A) ΔSm = ΔU/T = 9 kcal mol-1 / 314 K = 29 cal K-1 mol-1 = 119.9 J K-1 mol-1
(B) ΔSm(1 mol CH2-CH2) = ΔSm x 1 mol / 32 = 3.75 J K-1 = k ln(W2/W1)
W2/W1 = exp(3.75 J K-1 / 1.38x10-23 J K-1) = exp(2.7x1023) = 101.2x10^23