Introduction-1

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Transcript Introduction-1

Physics 307/607
Biology 307/607
Regular class times: MWF 10-10:50 AM
http://www.wfu.edu/~shapiro/biophysics12/
Instructors:
(1) Professor Martin Guthold, Phone: 758-4977, Office: 302 Olin, email:[email protected],
http://www.wfu.edu/~gutholdm/
(2) Professor Kim-Shapiro, Phone: 758-4993, Office: 208 Olin, email: [email protected],
http://www.wfu.edu/~shapiro/
Office hours:
Guthold: M, W, F; 1:30 pm – 2:30 pm, and by appointment.
Kim-Shapiro: M, W; 2:15 pm -4:00 pm, and 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 307/607
Biology 307/607
Syllabus
Grading:
Undergraduate Students:
2 Midterm exams...........................40%
Project………………………………10%
Final Exam.....................................30 %
Problem Sets..................................20%
Graduate Students:
2 Midterm exams........................... 30%
Project……………………………….10%
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 particular topic in biophysics. The
project could be a service learning project (see instructors for more information on that).
Project topic is due in two weeks (Feb. 1)
Project outline is due before spring break (March 9)
Complete project due last day of class (May 2)
** Graduate students need to do a 5-10 minutes presentation on one of the journal articles that are part of
the reading assignments (see reading list); or another article relevant to a lecture topic.
Physics 307/607
Biology 307/607
Syllabus
Exam Schedule:
Midterm 1: Monday, Feb. 27 (in-class)
Midterm 2: Friday, April 20 (in-class)
Final Exam: Monday, May 9, (9:00 am – 12:00 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 307/607
Biology 307/607
Syllabus
Tentative Syllabus:
Part I Biophysical Methods
1.
Introduction (Guthold) (~6 lectures)
1.1 Biological Macromolecules; 1.2 Molecular interactions; 1.3 Overview of Thermodynamics
Reading: van Holde, chapters 1-4 (partial).
2.
X-ray diffraction, DNA Structure (Guthold) (~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 307/607
Biology 307/607
Syllabus
Tentative Syllabus (cont.)
5.
Emission Spectroscopy (Guthold) (~4 lectures)
Reading: van Holde, Chapter 11
6.
Single Molecule biophysics (Guthold) (~3 lectures)
Reading: Chapter 16
7.
Electron Paramagnetic Resonance, Protein Function - Hemolgobin (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 Wednesday, Jan. 25):
1.
2.
3.
4.
5.
What is the Central Dogma of Molecular Biology (describe, sketch in your own words)?
Van Holde 1.2
(amino acid structure)
Van Holde 1.4
(amino acid structure)
Van Holde 1.7
(DNA structure)
Protein data bank exercises
(see 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
http://www.wfu.edu/~shapiro/biophysics12/
Bovine pulmonary
artery endothelial
cell
(image: Justin Sigley, WFU
Physics)
Introduction-1
Structures of biological Macromolecules
• In this course we will mainly deal with:
proteins,
nucleic acids, and membranes
(e.g. DNA, RNA)
From Voet & Voet Biochemistry
(e.g cell walls)
• Physical methods to examine the structure
and function of these biological molecules
Introduction-1
Structures of biological Macromolecules
Outline
• Central Dogma, Replication, Transcription, Translation
• Genetic code, DNA/RNA codons
• Nucleic acids, DNA, RNA
• DNA structure, twist, rise, linking number
• Amino acids, proteins
• Protein structure, 1o,2o, 3o, 4o structure
• Properties of amino acids, (small, large, neutral, charged, hydrophobic, hydrophilic, etc.)
• Protein data bank (PDB)
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).
Central dogma of Molecular Biology
Describes how the genetic information encoded (stored) in the ‘letter sequence’ of DNA is first transcribed and
then translated into an amino acid sequence, i.e. into proteins. (Crick, F.H.C. (1958): On Protein Synthesis. Symp. Soc. Exp. Biol.
XII, 139-163.Crick, F. H. C. (1970): Central Dogma of Molecular Biology. Nature 227, 561-563.)
Replication
(DNA polymerase)
Transcription
(RNA polymerase)
Genomic
DNA
mRNA
Protein
(Enzymes catalyze reactions in organism)
(Proteins – building blocks of organism)
The genome, or genomic DNA (deoxyribonucleic acids), of
an organism consists of a very long sequence of four
different nucleotides with bases A, C, G, T. Genomic DNA is
a double-stranded helix comprised of two complementary
strands, held together by A-T and C-G base pairs. The entire
genome is replicated by DNA polymerases (a protein) and
passed on to daughter cells during cell division. The genome
consists of many (usually thousands) of genes. A gene is a
specific, defined nucleic acid sequence that encodes one
particular protein. The human genome consists of about
3·109 base pairs and only about 30,000 genes (in higher
organisms, large parts the genome (80 – 98%) do not
encode any known proteins).
Transcription: RNA polymerase (a protein) binds to the
beginning of one particular gene and synthesizes an exact
RNA copy of that gene. RNA (ribonucleic acid) consists of
nucleotides with bases A, C, G, U. It is single-stranded.
Transcription stops at the end of each gene and the RNA
chain is released. A gene is on the order of a thousand
bases.
Translation: The RNA is moved to the ribosome. The
ribosome reads the RNA sequence (with the help of t-RNA)
and synthesizes an amino acid chain (polypeptide). The
polypeptide folds into a three-dimensional structure – a
protein (or part of a protein). There are 20 different amino
acids, thus three RNA letters are needed to code for one
amino acid. These triplets of RNA letters are called codons.
Eukaryotic cell
Central dogma
Picture in prokaryotic (bacterial)
cell and eukaryotic (higher) cell
Prokaryotic cell (no nucleus)
The human genome has about 30,000 genes (and
lots of non-coding DNA)
Simply speaking: one gene  one polypeptide
The sequence of bases in DNA codes for the sequence of amino acids in proteins
Transcription (making RNA from a DNA template):
RNA polymerase binds at a promoter (beginning of a gene), unwinds
DNA, and starts synthesis of an RNA copy of the gene
First real-time movies of a transcribing RNA polymerase 1,2
1.
2.
S. Kasas et al., Biochemistry 36, 461 (1997). (see Fig. 16.6 of book)
M. Guthold et al., Biophysical Journal 77, 2284 (Oct, 1999).
Kasas movie
http://www.youtube.com/watch?v=ZDH8sWiUsAM
http://www.youtube.com/watch?v=YEzRz1jmqNA
Credit:
8 minute movie of inner workings of a cell
BioVisions, Harvard University
How to compact 2 meters of DNA into 2 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
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
This is also how DNA and RNA match up (hybridize) in the binding pocket of
RNA polymerase during transcription!!
Normal Watson-Crick base pairing
A bit of nucleic acid nomenclature
Base
Base plus ribose sugar
Nucleoside (RNA)
Base plus deoxy ribose sugar
Deoxy-nucleoside (DNA)
Base plus ribose sugar plus
phospate (nucleotide)*
Adenine (A)
Adenosine (A)
Deoxy-adenosine (dA)
Adenosine monophospate
(AMP)
Cytosine (C)
Cytidine (C)
Deoxy-cytidine (dC)
Cytidine monophospate
(CMP)
Guanine (G)
Guanosine (G)
Deoxy-guanosine (dG)
Guanosine monophospate
(GMP)
Thymine (T)
(Methyluridine, m5U)
Thymidine (dT)
m5UMP
Uracil (U)
Uridine (U)
Deoxy-urdine (dU)
Uridine monophosphate
(UMP)
* Can also have two or three phosphates, and de-oxy variety, too
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
The structure of
DNA and RNA
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)
A quick aside: 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
L=T+W
s = W/T
L, linking number: Number of times one edge of ribbon linked around other – topological
property  cannot change w/o cutting. (calculate by L = T + W)
T, twist = winding of Watson around Crick – integrated angle of twist/2p along length, not an
integer, necessarily (calculate by T = (number of base pairs/(base pairs/turn))
W, writhe = wrapping of ribbon axis around itself – noninteger, geometric property
Supercoiling (Writhe) important in vivo (most DNA is slightly negatively supercoiled).
s = superhelical density
Note: There are topoisomerases to convert topoisomers. They can ‘remove a knot’ by breaking double-stranded DNA and religating DNA. Mutated topoisomerases cause cancer.
Sample problem
A circular, plectonemic (‘braided’) helix of DNA is in the B
form and has a total of 1155 basepairs.
1. What is the twist of the DNA?
2. 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 DW, DT, DL, and Ds?
Central dogma …continued
mRNA … messenger RNA
tRNA … transfer RNA
mRNA
Translation (inside the
ribosome (with help of
tRNA):
Translation: making a
peptide using mRNA as
the coding template
(peptide synthesis)
Genetic Code (same in all organism)
UAU, UAC =
Tyrosine
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 organic solvent (or vapor) to water (more in introduction-3).
DGtransfer  - 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.
The structure
of proteins
• 1° structure: A polymer
with a unique amino
acid sequence.
• There are twenty
different amino acids
Charged amino acids
Source: Kyte J & Dootlittle, RF; J. Mol. Biol. 157, 110 (1982)
Negatively charged
Positively charged
Nonpolar (hydrophobic) amino acids, aromatic
The structure
of proteins
• 1° structure: A polymer
with a unique amino
acid sequence.
• There are twenty
different amino acids
Nonpolar (hydrophobic) amino acids, alkyl
Hydrophobic amino acids
Nonpolar (hydrophobic) amino acids
Source: Kyte J & Dootlittle, RF; J. Mol. Biol. 157, 110 (1982)
Polar amino acids
The structure
of proteins
• 1° structure: A polymer
with a unique amino
acid sequence.
• There are twenty
different amino acids
Polar amino acids, disulfide with adjacent Cys
Polar amino acids, amines
Uncharged, polar amino
acids
Polar amino acids, aromatic
Source: Kyte J & Dootlittle, RF; J. Mol. Biol. 157, 110 (1982)
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
Example: Structure of Fibrinogen
(look at this structure in Protein Data Bank)
D nodule
E nodule
D nodule
a-hole
b-hole
Six polypeptide chains: 2 Aa (610 a.a.), 2 Bb (461 a.a.), and 2 g (411 a.a.) (human numbering).
Trinodular: 2 external D nodules; central E nodule (N-termini)
Parts not resolved: loopy C-a domain stretching back to E nodule (after residue 220), Nterminal of a- and b- chains (fibrinopeptides A and B) and N-terminal of g-chain (2x96
residues), C-terminal of g-chain(2x16 residues).
Dimensions: about 45 nm x 4.5 nm
17 disulfide bonds: within E nodule and braces at ends of the alpha helix coiled coils
Crystal structure of Chicken Fibrinogen (2x 1364 a.a.) . Z. Yang, J. M. Kollman, L. Pandi, R. F. Doolittle, Biochemistry 40,
12515-12523 (2001)
Formation of Fibrin Fibers
Fibrinogen
A
a
B
b
Thrombin
Fibrinopeptides A & B
Fibrin (protofibrils)
thrombi
n
fibrinoge
n
10 mm
SEM image (Hantgan) of fibrin clot
(plus platelets)
fibrin
Protofibril
formation
+
Lateral aggregation and
branching
10 mm
AFM image (Guthold) of fibrin clot
Further lateral aggregation
Image: M. Kaga, P. Arnold; Voet & Voet,
“Biochemisty”, Wiley & Sons, NewYork, 1990
The protein data bank
An Information Portal to Biological Macromolecular Structures
nearly 80,000 structures (Jan 2012)
Go to:
http://www.rcsb.org/pdb/home/home.do
We’ll do some exercises related to the
homework.