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Physics 303/607
Biology 303/607
Regular class times: MWF 10-10:50 AM
http://www.wfu.edu/~shapiro/biophysics10/
Instructors:
(1) Professor Kim-Shapiro, Phone: 758-4993, Office: 208 Olin, e-mail: [email protected],
http://www.wfu.edu/~shapiro/
(2) Professor Macosko, Phone: 758-4981, Office: 315 Olin, e-mail:[email protected],
http://www.wfu.edu/~macoskjc/
Office hours: Mondays and Wednesdays 2-4 pm; By appointment.
Texts:
1. Principles of Physical Biochemistry, by K.E. van Holde, W. C. Johnson, and P.S. Ho
2. Neurodynamix, by W.O. Friesen and J.A. Friesen.
3. Supplementary texts on reserve:
1. Biophysical Chemistry Part II, Techniques for the study of biological structure and
function, by Charles Cantor and Paul Schimmel (1980).
2. Biochemistry by Lupert Stryer (1988).
3. Additional reading will be assigned in the form of journal articles and handouts
Physics 303/607
Biology 303/607
Grading:
Undergraduate Students:
2 Midterm exams...........................40%
Project………………………………10%
Final Exam.....................................30 %
Problem Sets..................................20%
Graduate Students:
2 Midterm exams........................... 30%
Project……………………………….20%
Presentation of Journal Article.......10% **
Final Exam.....................................30 %
Problem Sets.................................20%
Emphasis in grading will be placed on how each problem is solved. All work showing how the solution was
obtained must be shown. An answer with the correct answer but poor method is inferior to one with the
wrong answer but good method.
Problem sets will generally be assigned for each chapter and the students will have one week to complete
them. Students may help each other on problem sets but each student must write their own solution to each
problem.
The project that all students do will be a 5-10 page paper focusing on a pparticular topic in biophysics. The
project could be a service learning project (see Dr. Kim-Shapiro for more information on that).
** Graduate students will present one of the journal articles that are part of the reading assignments (also
see reading list).
Physics 303/607
Biology 303/607
Exam Schedule:
Midterm 1: Friday, Feb. 29 (in-class)
Midterm 2: Friday, April 18 (in-class)
Final Exam: Friday, April 30, (2-5 PM)
Miscellaneous:
We will, at times, look at structures that are deposited in the protein data bank
(http://www.rcsb.org/pdb/home/home.do). The data bank contains the
coordinates of all solved protein, DNA, RNA and other bio-molecular structures,
usually to atomic resolution.
Physics 303/607
Biology 303/607
Tentative Syllabus:
Part I Biophysical Methods
1.
Introduction (Macosko) (~6 lectures)
Biological Macromolecules, Molecular interactions, overview of thermodynamics
Reading: van Holde, chapters 1-4 (partial).
2.
X-ray diffraction, DNA Structure (Macosko) (~5 lectures)
Fourier Transforms, Scattering, r(x) F(q), A helix , History of Watson and Cricks' discovery and its
implications
Reading: van Holde chapter 6, Watson and Crick Papers
3.
Light Scattering, Sedimenation, Gel Electrophoresis, Higher Order DNA Structure (Kim-Shapiro) (~4
lectures)
Sedimenation, mass spectrometry, Gel electrophoresis (Fick's Law), Light
Scattering (Classical, Dynamic, Polarized)
DNA Topology (Length, Twist, and Writhe), Chromosome Structure
Reading: van Holde, chapters 5 and 7, Polarized Light Scattering
4.
Absorption Spectroscopy, Protein Structure (Kim-Shapiro) (~4 lectures)
UV, VIS spectroscopy, linear and circular dichroism
Protein primary, secondary, tertiary, quaternary structure
Reading: van Holde chapters 8-10
Physics 303/607
Biology 303/607
Tentative Syllabus (cont.)
5.
Emission Spectroscopy (Macosko) (~4 lectures)
Reading: van Holde, Chapter 11
6.
Single Molecule biophysics (Macosko) (~3 lectures)
Reading: Chapter 16
7.
Electron Paramagnetic Resonance, Protein Function - Hemoglobin (Kim-Shapiro) (~4 lectures)
Electron Paramagnetic Resonance, Hemoglobin cooperativity Studies using EPR and time-resolved
absorption spectroscopy
Reading: Handout
Part II Membrane Biophysics
8.
Biological membranes and Transport (Kim-Shapiro) (~4 lectures)
Description of membranes, Diffusion, Facilitated transport, Nernst Equation, Donnan Equilibrium
Reading: van Holde chapters 13-14
9.
Nerve Excitation (Kim-Shapiro) (~3 lectures)
Neurons, Action Potential, Propagation of action potential, measurements in membrane biophysics,
Synaptic transmission
Reading: Frisens Sections 1 and 2
Introduction-1
Structures of biological Macromolecules
Homework (due Friday, Jan. 29):
1.
2.
3.
4.
5.
What is the Central Dogma of Molecular Biology?
Van Holde 1.2
(amino acid structure)
Van Holde 1.4
(amino acid structure)
Van Holde 1.7
(DNA structure)
Protein data bank exercises
(extra handout, protein, DNA structure)
Reading:
Van Holde, Chapter 1
Van Holde Chapter 3.1 to 3.3
Van Holde Chapter 2
(we’ll go through Chapters 1 and 3 first.)
Paper list (for presentations) is posted on web site
Introduction-1
Structures of biological Macromolecules
Outline:
1) What will we study?
2) Central Dogma (movie)
3) Structure of proteins & Genetic Code
Introduction-1
Structures of biological Macromolecules
• We will mainly deal with:
proteins,
nucleic acids,
(e.g. DNA, RNA)
and
.
membranes
From Voet & Voet Biochemistry
(e.g cell walls)
• Physical methods to examine the structure
and function of these biological molecules
Central dogma of Molecular Biology
(As spoken in the
language of biology,
i.e. narratives not
equations)
Transcription
(RNA polymerase)
Genomic
DNA
Reverse Transcription
mRNA
(reverse transcriptase)
Protein
(Enzymes catalyze reactions in organism)
(Proteins – building blocks of organism)
Biological Macromolecules – General Prinicples
- Well-defined stoichiometry & geometry. Not readily broken into tiny pieces
- Monomer is the building block (amino acid→proteins, nucleic acid→DNA/RNA)
(Macro = large. Up to ~ 25 residues = oligomer; >25 polymer)
• 1° structure: one-dimensional sequence
• 2° structure: local arrangement (a-helices, b-sheets, turns)
→super secondary structures: hairpins, corners, a-b-a motifs, etc.
• 3° structure: 3-D structure (e.g. folded protein), stabilized by H-bond,
hydrophobic forces, van-der-Waals, charge-charge, etc
• 4° structure: Arrangement of subunits (e.g. hemoglobin)
- Configuration vs. Conformation:
• Configuration – Defined by chemical (covalent bonds), must break bond to
change configuration (e.g. L-amino acid, D-amino acid)
• Conformation – Spatial arrangement (e.g. an amino acid polymer can have
a huge number of different conformations, one of which is the natively
folded protein).
Important Molecular interactions in
Biomolecules
The structure of proteins
1° structure: Amino acid sequence
– Twenty amino acids common to all organisms.
– Each has amino group, carboxyl group, R group and a hydrogen in
tetrahedral symmetry. Almost all organisms have “L” chirality, but some
virus have the mirror-image “D” chirality. (see board)
– Linked together by peptide bond. Peptide bond can be trans or cis.
– Proteins have prosthetic groups (e.g. heme) and amino acids can get
modified (sugars, phosphates, etc).
– Two important angles: Φ: N-Ca bond, Ψ: C-Ca bond  Ramachandran
plot of allowed angles (dis-allowed due to steric hindrance).
The structure of proteins
1° structure: Amino acid sequence
– Given N amino acids, there are 20N different sequences. Sequence
determines structure. If >20% homologous, probably similar structure.
Converse not true: very different sequences can have similar structures.
– Hydrophobicity/hydrophilicity values [or “hydropathy” values, i.e. “strong
feeling about”] determines protein folding. In aqueous environment, the
core is hydrophobic, the surface is hydrophilic; in the membrane, both
are hydrophobic.
– Kyte-Doolittle Scale – measure of hydrophobicity. Hydrophobicity is
determined by measuring the energy DGtrans of transfering an amino acid
from water to an organic solvent.
DG trasnfer  RT  ln P , where P 
 aq
 nonaq
,   mole fraction
• If DGtrans is positive – hydrophobic; if negative hydrophilic.
– There are charged and uncharged side chains. Proteins have net charge
and pockets of positive and negative charges, salt bridges. Isoelectric
point: pH where net charge of protein is 0.
Central dogma revisited
Transcription
(RNA polymerase)
Genomic
DNA
mRNA
Genetic Code
Protein
Genetic Code
(these tables are just a piece of the “Genetic Code”)
The G-ball:
A new way to explore the Genetic Code
Key:
Starred residues use class-II
(3’-OH charging, dimeric)
aminoacyl synthetases.
Residues in italics are
charged (white: positive,
black: negative).
Size of font corresponds to
residue size (and using all
lowercase for smaller than
average, all uppercase for
larger than average, first
letter uppercase for average).
The structure
of proteins
•
•
Negatively charged
1° structure: A polymer with
a unique amino acid
sequence.
There are twenty different
amino acids
-3
-2.6
Ala
Arg
4
Asn
Asp
2
Positively charged
Cys
Gln
0
1
2
3
4
5
-4.6
His
Ile
-2
Charged amino acids
-4
6
Glu
Gly
Leu
Lys
Met
-7.5
Phe
Pro
-6
-8
Ser
Thr
Trp
Tyr
Val
-1.7
Nonpolar (hydrophobic) amino acids, aromatic
The structure
of proteins
1.0
2.5
• 1° structure: A polymer
with a unique amino
acid sequence.
• There are twenty
different amino acids
Nonpolar (hydrophobic) amino acids, alkyl
1.0
2.3
2.2
Hydrophobic amino acids
3.1
-0.29
Nonpolar (hydrophobic) amino acids
1.1
Polar amino acids
The structure
of proteins
0.67
-0.75
• 1° structure: A polymer
with a unique amino
acid sequence.
• There are twenty
different amino acids
-1.1
Polar amino acids, disulfide with adjacent Cys
0.17
Polar amino acids, amines
Uncharged, polar amino
acids
-2.7
-2.9
Polar amino acids, aromatic
0.08
a-helix
(© by Irvine Geis)
The structure of
proteins
Biochemistry Voet & Voet
2° structure: alpha helix
Alpha helix:
- right-handed helix
- 0.15 nm translation (rise)
- 100° rotation (twist)
- 3.6 residues/turn
- Pitch: 0.54 nm
- stabilized by H-bonds
between NH and CO group
(four residues up).
Red – oxygen
Black – carbon
Blue – nitrogen
Purple – R-group
White – Ca
Hydrogen-bonds between C-O of nth
and N-H group of n+4th residue.
The structure
of proteins
2° structure: beta strand
Beta sheet:
- Can have parallel
and anti-parallel
- Distance between
residues: 0.35 nm
- H-bonds between
NH and CO groups of
adjacent strands
stabilized structure.
Note: Color-in atoms for practice
The structure of proteins
Higher Order Structure:
Super secondary (+2°) structure: b
turns, b-Hairpin, Greek Key, a-a,
bab, b barrel
H-bonding disfavored in aqueous
environment  b-sheets inside
globular proteins (prions: a-helix b
sheet)
Domains (are to 3° structure as sheets and helices are to +2° structure):
Structurally or functionally defined, eg calmodulin, DNA binding domain
3° Structure: Overall 3-D structure
Next time: pictures of peptide chains in fibrinogen molecule
Use sphere, ball and stick, ribbon representation
4° Structure
Non-covalently linked 3° Structures (eg Hemoglobin )
Homodimer vs hetero dimer, Hemoglobin is a heterotetramer
Dany’s lecture starts here
Outline for Friday January 15, 2010
• Introductions – How much Biology,
Physics, Chemistry have you had
•Web Page incl. Service learning
projects, HW etc
•Review
•Motivating question
•Nucleic acids
Introduction-1
Review.
• Central Dogma
• Primary, secondary, tertiary, quaternary
structure – PDB and VMD/Rasmol
• Molecular Interactions
• Amino acids, peptide bonds, angles
 aq


D
G

RT

ln
P
,
where
P

,   mole fraction
• Kyte Dolittle trasnfer
 nonaq
• Genetic Code
More detail on Kyte-Doolittle
DG trasnfer  RT  ln P , where P 
•
•
•
•
 aq
 nonaq
,   mole fraction
They used water to vapor, others have used water to ethanol.
If aq > nonaq then DG is positive – hydrophilic
If nonaq > aq then DG is negative – hydrophobic
e.g. DGtransfer for val is –2.78, DGtransfer for glu is +8.59 (in
Kcal/mole)
• Kyte and Doolittle actually used combnation of (1) –0.69
DGtransfer + 2.32 (2) (48.1)(fraction 100% buried) – 4.5 (3)
(16.45)(fraction 95% buried). They combined these three
things to get a hydropathy index.
• Now hydrophobic is positive and hydrophilic is negative.
Questions
Consider the DNA from a single white blood
cell of yours.
a) If you were to stretch it all out, how long
would it be?
b) Is the DNA different from that cell than
that from one of your endothelial cells?
c) Is the DNA from your white blood cell
different from that from the person sitting
next to you?
The structure of
DNA and RNA
•
•
•
Four monomer
building blocks
RNA has
ribose instead
of 2’deoxyribose
RNA has
Uridine instead
of Thymidine
Stabilizing factors in double-stranded (ds)-DNA
cruciform
Triple-strand
B-DNA:
A-DNA:
Z-DNA:
- right-handed
- right-handed
- left-handed
- most common form
- broader than B
- zig-zaggy
- 0.34 nm rise
- 0.26 nm rise
- ~12 bp per turn
-10.5 bp per turn
- ~11 bp per turn
- 3.4 nm pitch
- 2.8 nm pitch
- adopted sometimes by
(CG)n repeats.
- adopted in aqueous
- adopted in non-aqueous
- most common form for RNA
- has “hole” down the center
RNA molecules are more variable and can adopt structures that resemble
proteins (e.g. t-RNA below).
Aptamers are DNA and RNA molecules that fold into a 3D structure and
bind substrates (much like proteins)
What are aptamers?
Aptamers (from apt: fitted, suited; Latin aptus: fastened)
• Oligonucleotides which have a demonstrated capability to
specifically bind molecular targets with high affinity (KD = 10-6 to
10-9 M).
• First described by Joyce1 (1989), Tuerk & Gold2 and Ellington &
Szostak2 (1990).
• Binding properties depend on 3D structure and thus on sequence.
1 G.
F. Joyce Gene 82: 83-87 (1989)
2C.
Tuerk & L. Gold, Science 249, 505 (1990).
3A.
D. Ellington & J. W. Szostak, Nature, vol. 346, pp. 818-822, 1990
Three-dimensional solution structure of the thrombinbinding DNA aptamer d(GGTTGGTGTGGTTGG)
that we are working with (initially).
Twist, rise and
linking number
in DNA
Lk = Tw + Wr
s = Wr/Tw
Lk = linking number: Number of times one edge of ribbon linked around other – topological
property  cannot change w/o cutting. (calculate by Lk = Tw+Wr)
Twist = winding of Watson around Crick – integrated angle of twist/2p along length, not an
integer, necessarily (calculate by Tw = (number of base pairs/(base pairs/turn))
Writhe = wrapping of ribbon axis around itself – noninteger, geometric property
Supercoiling (Writhe) important in vivo (most DNA is slightly negatively supercoiled).
There are topoisomerases to convert topoisomers
Problem
A plectonemic helix of DNA is in the B form
and has a total of 1155 basepairs.
• What is the twist of the DNA?
• The DNA has a superhelical density of
-0.273. The DNA is put into an alcohol
solution and it takes the A form. What is
the DWr, DTw, DLk, and Ds?
Compact 2 meters of DNA into mm-sized nucleus?
(like folding a 1000 km long long fishing line (1 mm diameter) into 1m sized ball)
Nucleosome
http://www.rit.edu/~gtfsbi/IntroBiol/images/CH09/figure-09-07.jpg