MCB 65

Physical Biochemistry:
Understanding Macromolecular
Machines

Rachelle Gaudet
Martin Samuels
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What do we need to know to “understand”
macromolecular machines?

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What do we need to know to “understand”
macromolecular machines?

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 structure

energetics

structural methods

energetics
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 energetics
folding
ligand binding

protein-protein interactions

reactions

diffusion
membranes
metabolism
regulatory networks

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 Teaching staff

Instructors: Rachelle Gaudet (gaudet@mcb.harvard.edu)

Office hours: Northwest Labs 311.13 Tuesdays 6‐7PM

Preceptor Martin Samuels (msamuels@fas.harvard.edu)

Office hours: Fairchild 195 Fridays 3‐5 PM

Teaching Fellows: Enrique Garcia‐Rivera (egarciarivera@fas.harvard.edu)

Office hours: Fridays 11 AM – 12 PM
Emily Ricq (elricq@fas.harvard.edu)
Office hours: Mondays 11 AM – 12 PM

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 Course Objective
• Become familiar and conversant in the physical properties
and behaviors of biological molecules, with an emphasis on
macromolecules

• Three dimensional structure and organization

• Energy – classical & statistical thermodynamics
• Equilibria – driving forces for reactions
• Kinetics of reactions
• Integration of the concepts

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 Logistics
• Lectures:
• Mondays, Wednesday and Fridays
• 10:00am‐11:00am in NWL B‐109

• Sections:
• One mandatory combined discussion/laboratory session per week
• Monday noon – 2:30PM or 6 – 8:30PM in NW room B‐133
• Must make ONE of these times to be able to take the class

• Prerequisites:
• LPSA or LS 1a, Chem 20 or 17, and Math 1b MCB65
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Transporter associated with antigen processing (TAP)

Hydrolysis‐
dependent NBD peptide
opening
ER lumen

TAP1 TAP2

cytoplasm
ATP ATP
ATP ATP

ATP‐dependent
NBD closure

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 MCB 65 Lab and Section

Week Lab topic

1‐25‐16 no section
2‐1‐16 Journal club: TAP NBD structure paper
2‐8‐16 PyMOL tutorial/selecting TAP NBD mutations
2‐15‐16 No section (Presidents’ Day)
2‐22‐16 Protein purification/ review for exam on the 25th
2‐29‐16 SDS‐PAGE analysis of protein purification
3‐7‐16 Dialysis
3‐14‐16 No section (spring break)
3‐21‐16 Bradford assay
3‐28‐16 Protein stability assay
4‐4‐16 Midterm exam review
4‐11‐16 ATPase assay I
4‐18‐16 ATPase assay II
4‐25‐16 Final exam review
5‐4‐16 Final lab report due (reading period)
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 PyMOL: Visualizing, analyzing and presenting
macromolecular structures
• Through the semester, you
will learn to use PyMOL to
examine and present
structures
• Tutorial in Section 2
• Recurring presence in LPSs
• PyMOL files used in lectures
available on Canvas
• Additional PyMOL resources
available on the LPS page

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 Assignments and Grading
• Pop questions (10 x 0.5): 5%
• 12 handed out in class – 10 best out of 12 count
• Due at the beginning of the following lecture

• Lab and Problem Sets (10 x 2): 20%

• Due on Wednesdays at the beginning of lecture, or before
• EXCEPT LPS1 which is due next Monday 2‐1‐16 First LPS due
• Final lab report 10% a week from
today!
• Midterms (1 x 3 + 2 x 16): 35%
• Monday 2‐8‐16 quiz on amino acids and nucleotides
• Wednesday 2‐24‐16 covers lectures 1‐12
• Wednesday 4‐6‐16 covers lectures 13‐22

• Final: 30%
• During Finals period 5‐11‐16 at 2pm
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 MCB 65 Collaboration Policy
Discussion and the exchange of ideas are essential to doing academic work. We have
designed a range of assignments and exercises for this course to both enhance and
test your knowledge, and each has a different collaboration policy.

LPS Assignments, Final Lab Report, and Pop Questions:

In this course, you are encouraged to consult with your classmates as you work on
assignments. However, after discussions with peers, make sure that you can work
through the problem yourself and ensure that any answers you submit for evaluation
are in your own words and the result of your own efforts. In addition, you must cite
books, articles, websites, lectures, etc that have helped you with your work using
appropriate citation practices. For more information on citation, you can consult the
Guide to Citing in the Life Sciences
Midterm and Final Exams:
The Midterm Exams will occur during regular class periods, and the Final Exam during
the exam period. All exams will be closed‐book and individual work ‐ the work should
be all your own.
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 Reading materials

Required text:
The Molecules of Life: Physical and Chemical Principles
by John Kuriyan, Boyana Komforti and David Wemmer

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 Reading materials II
• Supplemented with selected literature reviews
• Some optional, some required reading
• as detailed on page 4 of the syllabus
• All PDFs available on the course website

• Supplemental textbooks available on reserve at Cabot

(listed on the syllabus and on the website)
• Contain many problems/exercises useful for additional practice

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 Learning the MCB 65 material

• Best practice for learning the material

• Read – or at least skim – ahead of the lecture
• Keep up to date with the readings
• Practice by working through problems – problem sets, sections,
additional problems (previous years’ LPSs and exams, end of
chapter problems, other textbooks)
• Ask questions!! Take advantage of office hours!

• General guidelines for coursework: in college, expect ~3hrs

of work on your own for each 1hr of lecture
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Questions?

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 Lecture 1:
Space, energy and time
Reading:
Lecture 1, today: Chapter 1, section A
Lecture 2, Wednesday: Chapter 1: 1.10 – 1.15
Supplemental reading: Chapters 1 and 2 provide a good review
for materials that should already be familiar

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 Fundamental goal of physical biochemistry
• Understand – and predict – how macromolecules perform
their biological function
• Requires knowledge of the physical principles that underlie
macromolecular structure, interaction and function
Learning goals
• Develop a sense of scale for molecules and their transactions
• Three‐dimensional structure • Space
• Classical & statistical thermodynamics • Energy
• Chemical kinetics • Time
• Identify the different noncovalent interactions important for
macromolecules
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 Macromolecule structure provides a spatial
context to understand their function
DNA polymerase in action
• Macromolecules are 3D
objects of defined structure

• Noncovalent interactions
control
• Structure
• Conformational states
• Interactions

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Lorena Beese, Duke University 1/25/16 20
 Macromolecules are polymers of basic chemical
building blocks
• RNA, DNA and proteins are the three major types of macromolecules

• They are sequences of monomers that are linked into polymers

• This sequence determines the macromolecule’s informational and

operational capabilities

• DNA: sequence encodes information

• Coding sequence
• Binding sites for transcriptional regulators
• Protein and RNA: sequence encodes 3D structure
• Folded entities perform biological reactions
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 There are multiple levels of protein structure
• Molecular biologists will often refer to the “structure of the protein”
and show a diagram similar to:
tyrosine kinase Src Y 416 Y 527
Myr SH3 SH2 Kinase

• However, biochemists and structural biologists have a different

picture in mind:

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 The levels of protein structure
• Primary structure
• Linear covalent chain of amino acids (from the 20
naturally available amino acids)
• Chemical structure of the protein chain
• Corresponds to the linear translation, using the
genetic code, of the gene’s DNA sequence

• Secondary structure
• Local conformation (i.e. noncovalent structure) of
a polypeptide chain stabilized by hydrogen bonds
• Building blocks of the three‐dimensional protein
structure
• ‐helix, ‐sheet, loop MCB65
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 The levels of protein structure
• Tertiary structure
• The folding of secondary structure elements
into a defined three‐dimensional arrangement
• Corresponds to the protein’s three‐
dimensional structure

• Quaternary structure
• Protein‐protein interactions leading to
noncovalent complexes of at least two
polypeptide chains
• Corresponds to the three‐dimensional
arrangement of multiple subunits into a
functional biological unit MCB65
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Four levels of protein structure
organization

PyMOL representations
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 Most atom types have standard colors

• Carbon (variable color)

• Nitrogen
• Oxygen
• Sulfur
• Phosphate or(often
Phosphate
omitted)

Molecular representations:
Sticks – show details of a structure
Cartoon – or ribbon diagram shows secondary structure
Spheres – details and packing, especially for small ligands
Surfaces – what other molecules can interact with MCB65
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 Packing inside proteins is very efficient

• Close packing of spheres:


 0.74074
3 2
• ~74% of volume occupied by atoms

• Organic liquids: ~45% of volume occupied by atoms

• Water: ~36% of volume occupied by atoms

• Proteins: ~70‐75%
• How do they do it? Complex puzzle!
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Proteins have a defined structure

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 Quote of note
• John Kendrew, on the structure of myoglobin, which he
solved in 1958:

• “Perhaps the most remarkable features of the molecule are its

complexity and its lack of symmetry. The arrangement seems to
be almost totally lacking in the kind of regularities which one
instinctively anticipates, and it is more complicated than has
been predicted by any theory of protein structure”

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Figure from The Molecules of Life (© Garland Science 2008)
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 Noncovalent forces shape structure
• Covalent interactions
• Chemical or primary structure

• Noncovalent interactions
• van der Waals interactions
• Hydrogen bonds
• Ionic interactions (salt bridges)

*** Both with self (intramolecular) and with surrounding solvent

(intermolecular)
• Hydrophobic effect
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Figure from The Molecules of Life (© Garland Science 2008)
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 van der Waals interactions

• All atoms can form van der Waals interactions

• Weakest of noncovalent interactions
• Over very short distances ~3‐4 Å
What does 1 Å
correspond to?
• Induced dipoles:

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Figure from The Molecules of Life (© Garland Science 2008)
 Hydrogen bonds

Hydrogen bond between

Dipoles – partial charges Aligned dipoles

~2.4‐3.0 Å

Dipole is described as a vector between the two charges held

apart, going from the negative charge to the positive charge.
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Figure from The Molecules of Life (© Garland Science 2008) 1/25/16 32
 Salt bridges (Ion pairs)
• Strongest noncovalent
interaction

• Electrostatic interaction is long

range (compared to van der
Waals and hydrogen bonds
with short effective distances)

• Salt bridge is an electrostatic

interaction with the charged
atoms close together
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Figure from The Molecules of Life (© Garland Science 2008)
Noncovalent interactions
• van der Waals
• ~ 1‐2 kJ/mol

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Noncovalent interactions
• van der Waals
• ~ 1‐2 kJ/mol
• Ion pair
• ~10‐100 x vdw
• ~10‐30 kJ/mol
depending on
environment

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Noncovalent interactions
• van der Waals
• ~ 1‐2 kJ/mol
• Ion pair
• ~10‐100 x vdw
• ~10‐30 kJ/mol
depending on
environment
• Hydrogen bond
• ~10‐20 x vdw
• Less (~5 kJ/mol) in H20

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Noncovalent interactions are readily made and
broken at physiological temperatures

Covalent
bonds
~1000 kJ/mol
Increasing energy (E)

~10‐30 kJ/mol

Noncovalent
interactions
Energy available
~5 kJ/mol from collisions
~ kBT ~ and vibrations at
~1‐2 kJ/mol physiological
temperatures

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 The hydrophobic effect drives folding

• The “hydrophobic effect” is a collective property of solvent

and solute molecules

• Hydrophobic molecules tend to cluster together to minimize their

surface area exposed to water
• Maximize the favorable interactions of water and hydrophilic
molecules together
• Most important determinant
of protein structure

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Figure from The Molecules of Life (© Garland Science 2008)
 F0F1 ATP synthase runs at 6000 rpm

Credits: John Walker MCB65

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F0F1 ATP synthase

• Whole assembly is
~0.5 MDa

~90 Å or 9 nm
• Turnover rate
• ~300 ATP / s
• ~100 revolutions / s
• ~3 revolutions / ms

• Each ATP stores ~50

kJ/mol
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 Some concepts to remember
• Protein structures have a hierarchy

• Noncovalent interactions are crucial to macromolecule

structure and interactions and range in energy
• van der Waals
• Hydrogen bond
• Ion pair/salt bridge

• Macromolecular functions are spread over a wide range of

time scales
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