Introduction to Biochemistry Chem-527_011
(Spring 2006)
Instructor: Colin Thorpe
204
Drake Hall
Phone:
831-2689
EMail:
cthorpe@udel.edu
Office Hours: Trial …
Tuesday 6-7 PM 204 Drake Hall.
Lectures: 4:30-5:45 PM; Memorial 111
Examinations:
Examination Date % of Grade
Exam 1
Thur, March 9 30
Exam 2 Thur,
April 20 30
Final To be announced (Final's
week) 40
There will be no make-up examinations. Medical excuses require a note from your
doctor (for excused absences on Exams 1 and 2, your score on the final will be
prorated to cover that 30% of your grade).
Text: Lehninger
Principles of Biochemistry 3rd. Edition (Nelson, D.L. and Cox, M.M.). Worth Publishers. ISBN: 1-57259-153-6
Visit the
publisher’s web site at: www:worthpublishers.com/lehninger
for useful
information and links
A tentative outline of
major topics is listed on the next page.
Please note that we cannot cover the whole of the book. I will direct you to what I think is
important as we progress. In the past my
tests usually come from, or are inspired by, material covered in class. I
anticipate that coming to class will be advantageous.
Introduction to Biochemistry Chem-527_011
(Spring 2006)
Tentative reading order
Chapters 1-3: An introduction to molecular
logic, cells and biomolecules (a review of some basic chemical concepts). These
are support chapters for background and/or review.
Chapter 4: Water
Chapters 5-7: Amino acids through protein
structure and function
Chapter 8: Enzymes
Chapter 9: Carbohydrates
Chapter 10: Nucleic acids
Chapters 11-12 Lipids
and Membranes
Chapter 14 Bioenergetics
Chapter 15 Glycolysis
Chapter 16 Citric
acid cycle
Chapter 17 Fatty
acid oxidation
Chapter 19 Oxidative
phosphorylation
Chapters 20-22 Some
topics in biosynthesis
Chapter 24 Genes
and Chromosomes
Chapters 25-29 Topics
in information pathways and recombinant DNA technology
Grading: The
distribution of grades averaged over the last few years:
(A/A-) totalled 26%; (all B grades) 35%; (all Cs) 33%;
(all Ds and Fs) 6%. The average grade
on the three exams was 56% (highest avg. grade was 95%). Please consider these statistics in judging
your prospects. A part of each exam
tests the application of concepts we cover in the course to material you may
never have encountered. The course is
graded on the curve and, given a comparable class, we expect a similar distribution
of grades. To give you an idea what to
expect a sample of exams is included in this packet. Note that metabolic charts are (and will be)
included in exams 2 and 3. Please note
that biochemistry is “CHEMISTRY brought to life” and there are
chemistry prerequisites for this course.
Extra stuff: There
are a few extra problems in the packet to reinforce some key topics. As noted above, there are also sample exams
in the packet … I will post the answers on the WEB, together with one set
of extra exams, later.
Finally: Good luck.
________________________________________________________________________________________
ANSWERS TO EXAMS IN THE PACKET – please
give them a go first before checking the answers:
FIRST CHEM527 (CT) EXAM 2004 answers __CHEM 527 First Exam F04.pdf
SECOND CHEM527 (CT) EXAM 2004 answers __CHEM 527 Second Exam F04.pdf
FINAL CHEM527 (CT) EXAM 2004 answers __CHEM 527 Final Exam F04.pdf
==============================================================================
EXTRA EXAMS
FIRST CHEM527 (CT) EXAM 2002 CHEM 527 Exam 1 F02.pdf
SECOND CHEM527 (CT) EXAM 2002 CHEM 527 Exam 1 Key F02.pdf
FINAL CHEM527 (CT) EXAM 2002 CHEM 527 Final F02.pdf
FIRST CHEM527 (CT) EXAM 2002 answers CHEM 527 Exam 1 Key F02.pdf
SECOND CHEM527 (CT) EXAM 2002 answers CHEM 527 Exam 2 Key F02.pdf
FINAL CHEM527 (CT) EXAM 2002 answers CHEM 527 Final Key
F02.pdf
==============================================================================
ANNOUNCEMENTS
IN CHRONOLOGICAL ORDER
Please
note the "no make up" exam policy announced above and in the packet
Office Hours: Tuesday 6-7 PM 204 Drake Hall.
2/13/06: Lehninger IV
edition placed "on reserve" in Morris library for CHEM527_011 (blue
and white book)
2/14/06:
Three additional exams posted +/- answer keys (see above in blue)
3/2/06: Exam 1 (March 9) coverage stops just before "Protein
Function". So it is up to and
including isoelectric focusing/2 D gels.
3/6/06 HELP SESSION for FIRST exam WED 8th 7-9 PM 210 BROWN LAB
For exam: please bring a calculator and a ruler
(or straight edge).
==============================================================================BRIEF
SYNOPSIS OF MATERIAL COVERED IN LECTURE
Reading advisory: Lehninger (L)IV Chapter 2.
(Lehninger III, Chapter 4) or any equivalent
2/7/06>>>
General introduction to the
course. Attention was drawn to the
typical grade distribution in CHEM527 taught by CT. Please take a look at the sample exams in the
packet to judge the style of exams and your prospects in the course.
We started with water (again). Concepts
polarized bond, dipole, H-bond, electrostatic bond, dielectric, strength of
typical covalent bond and "typical" H-bond, solvation, hydrophilic,
hydrophobic, clathrates, amphipathic, surfactants, soaps, micelles, bilayers,
biological membranes, monolayers, a molecular explanation of surface tension,
the importance of surfactants in physiology.
Then we started with equilibria (again)
ionization of water,
first and second order reactions with
reference to a general reaction: A ↔ B + C
ion product of water, pH scale
I'll assign suggested problems from
packet after next lecture.
==========================================================
2/9/06>>>
We reviewed the following topics
(again)
strong and weak acids/bases, the Henderson
Hasselbalch equation, titrations of a weak acid by a strong base, pH/titration
curves, buffers, the components of the buffering capacity of biological fluids,
the need for pH control, and the general effect of pH on the absorption of
drugs with titrating groups.
>>>problems from the handout and 2004
exam set:
Handout:
page 3: questions 1, 2
First exam 2004: questions
1a, b, c, d, e.
Final exam 2004: question 8 a, b, e.
First exam 2002 questions 1 a, b, c, d, e, f
Final exam 2002 questions 12 a, b
Started "amino acids, peptides and
proteins" section
Reading advisory: Lehninger IV
(pp. 75-88). Lehninger III, (pp.
113-129). In both editions, the
subsequent sections … on protein purification will be covered a little
later Don't need them just now. For comparative coverage to other texts you
can check what is covered on Lehninger IV (on reserve in Morris Library).
In lecture we discussed: a typical
amino acid; the peptide bond; the immense number of possible combinations of sequences
in a 20 residue peptide - considering 20 possible amino acids at each position
within the chain. We covered primary,
secondary, tertiary and quaternary structure; common classes of proteins
(enzymes, transport proteins, storage proteins, antibodies, receptors, toxins,
hormones and structural proteins).
===========================================================
for problems see end of this section
today we covered structures of amino acids – what you need to know (including 3- and 1-letter codes); ionization behavior of amino acids (including titration curves); charges on peptides and proteins; importance of amino acid sequence; invariant, conservative and non-conservative changes in homologous proteins (like a series of cytochrome c proteins from a range of different organisms). I described the use of sequences in deducing phylogenetic relationships (evolutionary trees). I mentioned how the amino acid sequence of a protein might give clues to its function and its 3-dimensional structure – the example I gave was of the discovery of a protein from chicken egg white that was purified in the Thorpe lab by a former graduate student. We got amino acid sequences of fragments of the protein that we prepared by digesting the protein with trypsin.
I showed one such peptide for an example:
Here is its sequence (you can copy and paste as described below)
SLYSPSDPLELLGADTAERRLLGSPSA
If you want to see what you can do with a peptide – interrogate the hundreds of thousands of protein sequences in the protein sequence databases with a few key strokes ……
1. Go to BLAST: http://www.ncbi.nih.gov/BLAST/
2. Under protein (at right) click on protein-protein (BLASTp)
3. In box paste the sequence: SLYSPSDPLELLGADTAERRLLGSPSA
4. Now click BLAST!
5. the screen will tell how long it will take (depends on time of day) – usually few seconds
6. then you should get a list of "significant hits"
[when we did this for the very first time we realized that our obscure little project on chicken eggs was going to be relevant to human health – how about human "bone derived growth factor"!]
(7. if you repeat this with a truncated version of the sequence e.g. SLYSPSDPLE BLAST says "no significant hits" WHY? – the answer is related to the question why would you never thinking of searching for the sequence SLY in the databases)
Suggested problems you should now be able to address. Try them first before peeking at the answers:
Packet page 3: Problems 3, 4, 5, 6
[if you need pKs they will be provided – here you could use the front page of First exam 2004]
2004 FIRST EXAM: Questions 2, 3, 6
2004 FINAL Questions 5,
2002 First exam Questions 2, a, b, c; Q. 3a; Q. 4, 5
2002 FINAL exam No Q for this lecture
==============================================
Reading
advisory: Lehninger IV (pp. 96-111).
Lehninger III, (pp. 137-150). In
both cases the details of mass spectroscopy sequencing will not be
needed – just the principles as outlined in class. Note, we do not cover peptide synthesis
(there are semi-automated ways to make defined peptides in the lab)
More
reading Lehninger IV Chapter 4: pp.
116-153; III: Chapt. 6
pp. 159-198. Note the level of
detail in lecture to judge relative importance of sections in readings.
In lecture today we re-discussed the
importance of protein sequence (primary structure); Edman sequencing and
its limitations; sequencing peptides by mass spectrometry and
limitations; disulfide bond reduction and cysteine alkylation
strategies. Enzymatic (trypsin and
chymotrypsin) and chemical (CNBr) methods for fragmenting proteins
into specific fragments was outlined (there are many other methods). The need for overlaps in assembling sequences
was introduced. As an alternative, the
deduction of protein sequences from DNA sequences was mentioned with the
limitation that this does not provide direct evidence for post-translational
modifications .
General aspects of protein folding were
introduced starting with the roles (or otherwise) of H-bonding, Van der
Waals, electrostatic and hydrophobic interactions in the net stabilization
of globular proteins in water compared to their unfolded states.
Lecture concluded with introduction to
the planarity of peptide bond and the existence of trans and cis peptide
conformers. We will next deal with
secondary structure
___________________________________________________
Suggested Questions:
Exam 1 (2004)
Q7, a, b and f (the other parts will require a bit more context –
see later)
Exam 1 (2002)
Q3 a and (after next Tuesday) b.
===================================================
Reading
advisory: Lehninger IV Chapter 4: pp.
116-153; III: Chapt. 6
pp. 159-198. Note the level of
detail in lecture to judge relative importance of sections in readings.
In
lecture today we discussed: phi and psi
angles, and their representation in graphical form for various types of
secondary structures. I forgot to call
them by their popularizer (Ramachandran Plots).
We dealt with disallowed regions of the plot with steric clashes. We covered the alpha helix and its
characteristics and stability, helical wheel representations and amphipathic
helices; turns; beta structure (parallel and antiparallel); and the collagen
triple helix. We stressed the importance
of GLY in the sequence, and a genetic disease associated with a GLY-SER
mutation (involving a dominant trait).
We discussed post-translational modifications in collagen (e.g.
hydroxyproline and the modifications involving lysine crosslinking).
In a
section concerning structural proteins we mentioned the archetypical ones:
keratin, fibroin and collagen.
Particular emphasis was placed on keratins. First we dealt with the concept of
pseudo-repeats abcdefg (with "a" and "d" being hydrophobic
– helping stabilize the coiled coil); we mentioned soft and hard keratins
and the correlations with cysteine (disulfide) content; we discussed the
arrangement of the intermediate filaments (coiled-coils) embedded in
disulfide-rich keratin associated proteins.
Permanent waving was mentioned as involving reversible reduction and
reoxidation of disulfide bridges.
Finally
we discussed briefly protein crystallography and solution and solid-state NMR
and their utility in determining the structures of proteins.
Tertiary
and Quaternary structures are next.
===================================================================================================================
Today
we started by showing a series of overheads of the structures of proteins. We discussed common structural motifs that
can lead to more complex globular proteins – including bundles of
helices, sheets, beta-alpha-beta structures, beta-turn-beta etc … and how some of these can form
barrels and etc.
The
concept of domain was introduced and examples of large multidomain
proteins were given. Quaternary
structure was illustrated - from the simplest dimer to the protein capsids
of viral particles. Denaturation
was next and the discussion included perturbants, denaturants and
unfolding profiles. Methods to
studying the refolding of proteins were mentioned with the likely time-scales
for refolding. The hierarchical
nature of protein folding was described and the slowest steps in folding
outlined. We mentioned that hyperbolic
term the "molten globule" and the Levinthal paradox. Snags in protein folding (e.g. aggregation)
were mentioned, along with the concept that large proteins often denature
irreversibility. The need for chaperones
and foldases in vivo was introduced.
The specialized role disulfide bonds play in the folding of certain extracellular
proteins was mentioned – disulfides were mentioned as stabilizing
individual polypeptide chains (intra-chain) or between chains
(inter-chain). The lecture concluded
with a brief mention of the minicollagens that are crosslinked by disulfide
bonds to harden the walls of the stinging organelle of jellyfish (so that it
can survive an internal hydrostatic pressure of >2000 psi). For certain proteins and materials,
disulfides are structurally critical.
(Next
lecture we start by reviewing Anfinsen's experiment with ribonuclease folding
and then we will discuss protein purification methods (to be found in Lehninger
IV 89-96 [or Lehninger III 130-137]
Next we will
cover "Protein Function" (talking about just the oxygen binding
'globins) NOTE: NOT FOR EXAM 1
Reading
Lehninger IV, 157-174) ; Lehninger III 203-221) NOTE: NOT FOR EXAM 1
===================================================================================================================
We
discussed Anfinsen's experiments with folding of reduced ribonuclease (RNase). The reaction between disulfides and a
chemical reductant such as beta-mercaptoethanol
(2-mercaptoethanol). The nucleophilic
attack of thiolate on disulfides was also discussed in terms of the enzyme protein
disulfide isomerase.
The
general strategy of protein purification was discussed first highlighting the
need for an assay for the specific protein of interest and a means to
determine total protein amounts more generally. The following protein purification techniques
were covered: ammonium sulfate
precipitations (salting out); separations on basis of size (gel fitration/size
exclusion chromatography and dialysis/ultrafiltration); separations on basis of charge (e.g. cation
and anion exchange chromatography); affinity chromatography - and
its more modern incarnations e.g. involving His-tags.
Next
we discussed techniques for evaluating whether protein is pure or not: SDS-PAGE under reducing or
non-reducing conditions. This section
will be continued
==============================================================================
Recap
SDS-PAGE with examples.
END OF EXAM 1 MATERIAL
NEW MATERIAL
NOT ON FIRST EXAM – starting with myoglobin and hemoglobin.
We
discussed ligand binding principles.
Association and dissociation equilibrium constants. The differences between plots of fractional
saturation vs. free ligand and total ligand. In terms of oxygen binding the various ways
"free oxygen" is manipulated.
Mentioned why oxygen binding proteins are generally necessary (the conc.
of dissolved oxygen in air equilibrated buffer is relatively small (0.24
mM)). Discussed a thought experiment
involving adding deoxy-myoglobin to a beaker of stirred buffer equilibrated
with air.
Started
talking about hemoglobin quaternary structure – to be continued.
===============================================================================