Interdisciplinary Biochemistry Graduate Program | Macromolecular Structure and Interaction
B503 | 1296 | Oakley, M.

1.	Description

Principles of inter- and intra-molecular interactions; structural
stability of proteins and nucleic acids; thermodynamic and kinetic
analysis of complex binding; experimental methods for analysis of
macromolecular structure and binding.

2.	Logistics
3 credit hours
Required for all Biochemistry graduate students

3. 	Prerequisites

B501 or undergraduate biochemistry (equivalent to C483 or C484), one
semester of undergraduate organic chemistry (C341 or equivalent) or
consent of instructor.  Undergraduate (bio)physical chemistry (C481
or C361 or equivalent) is strongly recommended.
Meets with Chemistry C581.

4.	Objectives

1.	To reiterate essential concepts in physical chemistry
2.	To familiarize students with the physical forces and effects
that control the structural features and stabilities of proteins,
DNA, and RNA and their complexes
3.	To familiarize students with the commonly used experimental
methods for probing the structures, stabilities and binding
interactions of macromolecules, including background theory,
information content, experimental methods, and the form and
interpretation of the data
4.	To familiarize students with rigorous, quantitative
approaches to distinguishing between modes of binding to determining
binding constants and other relevant parameters.
5.	To train students at identifying appropriate experiments
methods for solving specific biochemical problems.

5.	Grading

Homework (70%), including problems, sequence alignments and graphics
exercises, simulations, curve-fitting, experimental proposals, data
Group exercises (problem based learning) (20%)
Participation in class discussions (10%)

6.	Textbook


7.	Course topics

Part 1: Background Material (3 lectures)

1.	Biochemical problems: examples highlighting the need for
detailed structural and physical data, including the identification
of exactly what information is needed.
2.	Review of basic thermodynamic concepts: association and
dissociation constants, free energy, enthalpy, entropy, rate
constants, activation energy, pKa values
3.	Review of basic thermodynamic concepts continued

Part 2: Macromolecular Structure (8 lectures)

4.	Protein secondary structure: (example: prion proteins)
Ramachandran plots, statistical/thermodynamic propensities and
5.	Protein secondary structure: theory and practice of far UV
circular dichroism (CD) spectroscopy
6.	RNA: secondary structure prediction – theory and practice
(Homework exercises: PDB files and molecular graphics)
7.	Molecular mechanics and molecular dynamics simulations
8.	High resolution macromolecular structures – x-ray
9.	High resolution macromolecular structures – NMR spectroscopy
10.	Quaternary structure: size exclusion chromatography, light
11.	Quaternary structure: determination by sedimentation
analysis; Kd values by equilibrium sedimentation

Part 3: Macromolecular Stability (9 lectures)

12.	Fluorescence as a probe of tertiary structure: theory and
13.	Thermodynamics of folding: equilibrium unfolding (linear
extrapolation method; CD/fluorescence detection)
14.	Thermodynamics of folding: physical effects that stabilize
15.	Thermodynamics of folding: chemical and temperature
denaturation – physical basis
16.	Thermodynamics of folding: the role of electrostatics –
examples from Nick Pace’s work
17.	Thermodynamics of folding: differential scanning calorimetry,
enthalpy-entropy compensation, Cp
18.	The hydrophobic effect, Cp, relationship to buried
hydrophobic surface area, cold denaturation
19.	Thermodynamics of folding: mutational studies to probe forces
stabilizing structures – examples from Alan Fersht’s work
20.	-lytic protease: the native form is not necessarily the most
stable (David Agard’s work)

Part 4: Thermodynamics of Macromolecular Interactions (13 lectures)

21.	Modes of binding (1 lecture): single versus multiple site,
independent versus cooperative, etc.
(Homework exercise: simulating simple binding curves)
22.	Fitting simple (single site) binding (2 lectures):
establishing equilibrium conditions, graphs, equations, simulations,
curve fits, errors
(Homework exercises: fitting binding curves)
23.	Experimental methods for measuring binding: equilibrium
diffusion, UV, fluorescence, NMR
24.	Isothermal titration calorimetry
25.	Prediction of binding thermodynamics: molecular modeling;
Ernesto Freire’s semi-empirical approach
26.	Non-equilibrium methods: gel shift, radioligand binding, the
standard state problem
27.	Surface plasmon resonance: theory, practice, data analysis,
28.	Identification of binding sites: chemical methods (protease
protection, mutational analysis, protection interference, affinity
cleavage); mutagenesis
29.	Identification of binding sites: spectroscopic methods (NMR,
EPR, fluorescence) and x-ray crystallography
30.	Fitting more complex binding: multiple sites, multiple K¬d
values, cooperative binding
31.	Fitting more complex binding: continued
32.	Practical examples  - 2 site independent binding
33.	More practical examples – 4 site cooperative binding –
hemoglobin (Gary Ackers’ work)

Part 4: Kinetics and Dynamics of Macromolecular Interactions (4

34.	Measurement of binding and folding kinetics: mixing
35.	Hydrogen exchange of macromolecules: thermodynamic and
kinetic information
36.	Hierarchical protection in cytochrome c (Englander approach)
37.	Exchange phenomena: influence of binding or conformational
equilibria on the appearance of biophysical data; dependence on and
determination of exchange rates

Part 5: Experiment Selection and Design (6 lectures)

38-43	: Problem based learning: selection and design of experiments
to answer specific questions