Introduction to Biochemistry Chem-527 (Spring 2008)
http://www.udel.edu/chem/thorpe/Chem527/links527.html
exam #2 this Thursday
Help session Wed evening 7:30 PM ( not 7 PM)
7:30 – 9:30 PM in 101 BROWN LAB (or if occupied in one of the lecture rooms above it on the second floor of Brown)
Scope of exam: from tertiary structure through enzymes
Instructor: Colin Thorpe
204 Drake Hall
Phone: 831-2689
EMail: cthorpe@udel.edu
Office Hours: Monday 5:30 - 6:30 PM Vamsi Kodali (vamsi at udel.edu) 308 DRAKE HALL
Tuesday 5:50 PM – 7 PM CT (cthorpe at udel.edu) 204 DRAKE HALL
Friday 4:30 - 5:30 PM Vamsi Kodali (vamsi at udel.edu) 308 DRAKE HALL
Lectures: Tu/Th 4:30 PM - 5:45 PM Brown Lab Room 101
Examination Date % of Grade
Exam 1 Tuesday, March 4 25
Exam 2 Thursday, March 27 25
Exam 3 Tuesday, April 29 26
Exam 4 Tuesday, May 20 24
There will be no make-up examinations. Medical excuses require a note from your doctor (for excused absences on Exams 1-4, a comprehensive exam will be provided to be taken in the traditional final exams week. The grade on this exam will substitute for the missed exam).
Text: Lehninger Principles of Biochemistry 4th Edition (Nelson, D.L. and Cox, M.M.). Worth Publishers. ISBN: 0716743396
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.
Tentative reading order
Chapters 1: An introduction to molecular logic, cells and biomolecules (a review of some basic chemical concepts). This is a support chapters for background and/or review.
Chapter 2: Water
Chapters 3-5: Amino acids through protein structure and function
Chapter 6: Enzymes
Chapter 7: Carbohydrates
Chapters 10-11 Lipids and Membranes
Chapter 13 + Part II Bioenergetics
Chapter 14/15 Glycolysis
Chapter 16 Citric acid cycle
Chapter 17 Fatty acid oxidation
Chapter 19 Oxidative phosphorylation
Chapters 21-22 Some topics in biosynthesis
Chapter 24 + 8 Genes and Chromosomes
Chapters 9 Topics in information pathways and recombinant DNA technology
Grading: The distribution of grades averaged over the last few years:
(A/A-) totalled 25%; (all B grades) 34%; (all Cs) 35%; (all Ds and Fs) 6%. The average grade on the three exams was 58% (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 will be included on the Website. (Please note: the coverage between the exam will differ because of scheduling difficulties peculiar to this semester). Note that metabolic charts will be included where appropriate. Please note that biochemistry is “CHEMISTRY brought to life” and there are chemistry prerequisites for this course.
Extra stuff: I will post a brief synopsis on the Webpage together with readings and announcements and extra material as appropriate.
Finally: Good luck.
ANNOUNCEMENTS
FIRST help session Sunday March 2 at 7-9 PM in 101 BROWN LAB (or if occupied in one of the lecture rooms above it on the second floor of Brown)
SECOND help session Wed March 26 at 7-9 PM in 101 BROWN LAB (or if occupied in one of the lecture rooms above it on the second floor of Brown)
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FIRST EXAM INFORMATION
KEY PDFs\Chem 527 Exam 1 Key S08.pdf
AVERAGE 67.1
High = 98 Low = 6
Approximate advisory grades are given below to give you an INDICATION (with a margin of error of probably one +/- grade increment) should this exam be you entire grade for the course (it isn’t!).
A = 88 and up
A- = 84-87
B+ = 79-83
B = 69 -78
B- = 65-68
C+ = 61-64
C = 47-60
C- = 40-46
D+ = 38-39
D = 35-37
D- = 29-34
F = less than 29
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EXAM PDFs
Green highlighted entries are some old exams. Red .. are the keys. Please don’t peek at the keys until you need to!
Please note because of scheduling difficulties there will be 4 hourly exams (see schedule) and not final in finals week (as we have always done before … further explanation in class). Hence the material covered in each exam will be smaller. The coverage will be announced in good time. We don’t cover exactly the same material each time – so please keep track. Meanwhile here are some of the types of questions you may encounter.
FIRST CHEM527 EXAM 2006F PDFs\exam1_chem527_06F.pdf
FIRST CHEM527 EXAM 2006F PDFs\Chem527E1keyF06.pdf
SECOND CHEM527 EXAM 2006F PDFs\exam2_Chem527_06F.pdf
SECOND CHEM527 EXAM 2006F PDFs\CHEM 527 Second Ex_Key F06.pdf
FINAL CHEM527 EXAM 2006F PDFs\chem527_Final_ex_F2006.pdf
FINAL CHEM527 EXAM 2006F PDFs\CHEM527_06F_FinalKEY.pdf
FIRST CHEM527 (CT) EXAM 2004 PDFs\1stFall2004.pdf
FIRST CHEM527 (CT) EXAM 2004 PDFs\CHEM 527 First Exam F04.pdf
SECOND CHEM527 (CT) EXAM 2004 PDFs\2ndFall2004.pdf
SECOND CHEM527 (CT) EXAM 2004 PDFs\CHEM 527 Second Ex_Key F04.pdf
FINAL CHEM527 (CT) EXAM 2004 PDFs\FinalFall2004.pdf
FINAL CHEM527 (CT) EXAM 2004 PDFs\CHEM 527 Final Exam F04.pdf
FIRST CHEM527 (CT) EXAM 2002 PDFs/CHEM 527 Exam 1 F02.pdf
FIRST CHEM527 (CT) EXAM 2002 PDFs\CHEM 527 Exam 1 Key F02.pdf
SECOND CHEM527 (CT) EXAM 2002 PDFs\CHEM 527 Exam 2 F02.pdf
SECOND CHEM527 (CT) EXAM 2002 PDFs\CHEM 527 Exam 2 Key F02.pdf
FINAL CHEM527 (CT) EXAM 2002 PDFs/CHEM 527 Final F02.pdf
FINAL CHEM527 (CT) EXAM 2002 PDFs\CHEM 527 Final Key F02.pdf
Please note, as we have said already, that there are chemistry pre-requisites for this course – so dust off that introductory text and remind yourself how to do basic stuff such as moles/molar/C1*V1 etc / logs / pH / stoichiometry / equilibria / kinetics / basic organic / curved arrows / bonding and etc.
Biochemistry is bioCHEMISTRY
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BRIEF SYNOPSIS OF MATERIAL COVERED IN LECTURE
(to be completed after each lecture [hopefully])
>>> Reading advisory: Lehninger (L)IV Chapter 2 and Chapter 3 (pp. 75-88; the subsequent sections … on protein purification will be covered a little later Don't need them just now).
Please note: Chapter 1 is not specifically covered – just useful background/synopsis and review from earlier classes.
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 (above) 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, 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 (without surfactants breathing would be very difficult).
Then we started with equilibria (again) ionization of water [remember - we need to assume basic familiarity with prerequisite materials – here equilibria such as A ↔ B + C)]
We reminded you about: ion product of water and strong and weak acids/bases
Feb. 14 2008
>>> Problems Water – titrations and buffers
First exam 2006F: questions: 1a, b, c, e; q. 6
Final exam 2006F: questions: 8; 9c
First exam 2004: questions: 1a, b, c, d, e.
Final exam 2004: questions: 8 a, b, e.
First exam 2002 questions: 1 a, b, c, d, e, f
Final exam 2002 questions: 12 a, b
We started with 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. Started "amino acids, peptides and proteins" section. We covered structures of amino acids – what you need to know (including 3- and 1-letter codes for all 20-amino acids commonly found in proteins ). You need to know (e.g. the structures of G, A, F, C, D and K).
>>> Reading advisory: Lehninger (L)IV Chapter pp. 96-111 and for the next part dealing with 3D structure of proteins pp. 116-153
Feb 19 2008
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 mentioned how the amino acid sequence of a protein might give clues to its function – 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 (we will talk about trypsin next lecture).
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.nlm.nih.gov/blast/Blast.cgi
2. Under “Basic Blast” click protein blast
3. In box (“Enter accession number … or FASTA sequence) paste the sequence: SLYSPSDPLELLGADTAERRLLGSPSA
4. Now click BLAST (the blue button)
5. the screen will tell how long it will take (depends on time of day) – usually few seconds to a minute or so ….
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" – it was the first entry then and the 9th entry now!]
This should give you a feel for negotiating one of the most useful databases for protein primary structure similarities …
We then resumed discussion of the importance of protein sequence (primary structure); Edman sequencing and its limitations; sequencing peptides by mass spectrometry and limitations; and finally DNA sequencing as a means to get protein primary structure information (and the problem that it doesn’t usually indicate post-translational modifications).
>>Reading advisory Lehninger IV Chapter 4: pp. 116-153. Note the level of detail in lecture to judge relative importance of sections in readings. (note: after this reading … we will go back to address protein purification and characterization pp. 89-96; Lehninger IV)
In lecture we recapped the importance of the primary sequence. We discussed sequencing strategies; 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 .
We introduced the planarity of peptide bond and the existence of trans and cis peptide conformers. We discussed: phi and psi angles, and their representation in graphical form for various types of secondary structures (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. Next up …. More secondary structure …..
>>Suggested Questions:
Try them first before peeking at the answers:
[if you need exact pKs they will be provided – here you could use the front page of First exam 2004]
2006 FIRST EXAM: Questions 3, 4, 5a, b. Q7 a and b 5c,d,ef,g (I, iv, v)
2006 FINAL Questions 8
2004 FIRST EXAM: Questions 2, 3, 6. Q7, a, b and f (the other parts will require a bit more context – see later)
2004 FINAL Questions 5,
2002 First exam Questions 2, a, b, c, d; Q. 3a b; Q. 4, 5.
2002 FINAL exam 12d
We discussed 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. 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 (we drew attention to the misrepresentations of hard and soft-keratins in the textbooks). Permanent waving was mentioned as involving reversible reduction and reoxidation of disulfide bridges.
Some additional problems before the first exam:
2006 FIRST EXAM: Questions 5c,d,ef,g (i, iv, v); 7, 9
2004 FIRST EXAM: Q4, 5. 7a, b, f,
2002 FIRST EXAM: Q6, 7b, 8
<<<END OF EXAM ONE MATERIAL>>>
We next discussed briefly protein crystallography and solution and solid-state NMR and their utility in determining the structures of proteins. We showed a series of overheads of the structures of proteins. We discussed protein design principles and 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.
We next discussed briefly protein crystallography and solution and solid-state NMR and their utility in determining the structures of proteins. We showed a series of overheads of the structures of proteins. We discussed protein design principles and 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.
We discussed snags in protein folding (e.g. aggregation – formation of fibrils a mention of amyloid in diseases such as Alzhemier’s) , along with the need for chaperones and foldases in vivo. We mentioned that large proteins often denature irreversibility.
Finally the specialized role disulfide bonds play in the folding of certain extracellular proteins was duiscussed – disulfides were mentioned as stabilizing individual polypeptide chains (intra-chain) or between chains (inter-chain). 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).
A short treatment of how to purify proteins Lehninger IV, pp. 89-96
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). Next came separations on basis of charge (e.g. cation and anion exchange chromatography); and then the principles of 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. We discussed how the combination with molecular weight information from size exclusion chromatography can be used to get information about quaternary structure. We discussed isoelectric focusing of proteins and protein mixtures and described 2D methods that can resolve complicated protein mixtures for proteomics. Examples of how to combine SDS-PAGE data under reducing and non reducing conditions were given.
Sample questions:
2002 First 1g; 2e, 7; 9, 10a, e, f, g
2004 First 7, 8, 9a, d,
2006 First 1d, 2; 8
2002 Final 12e
2004 Final 8c
Myoglobin and hemoglobin
Reading will be Lehninger IV, 157-174
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.
We mentioned myoglobin structure, the heme group, coordination environment. Hemoglobin quaternary structure – sigmoidal binding behavior of oxygen. This positive cooperativity rationalized by two extreme models – the concerted and sequential models. The former is a convenient, workable, model for us. Using this concerted model we discussed the two-state "T to R" equilibrium and the modulation of this equil. by oxygen, carbon dioxide, protons, D(B)PG, carbon monoxide and etc. These linked equilibria are important in many phases of ligand binding. A number of these allosteric modulators of oxygen binding were discussed in some detail, including carbon dioxide, protons (Bohr effect), DPG and carbon monoxide. We discussed sickle cell disease. We also mentioned life without hemoglobin – ice fish and etc.
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After we have finished hemoglobin you should be able to tackle these questions:
2002 Second 1d, 6, 9
2004 Second 1c, 4, 8
2006 Second 2c, 2d, 3, 6
2002 Final 12c, 13,
2004 Final 7, 8i
2006 Final 9d, h
Some more Questions for Protein Function section:
2004: First exam 1g.
2004: Exam 2. 1c, 4, 8, 10i
2004: Final exam. 7, 8i,
2002: Second exam, 1d, 6, 9, 10h
2002, Final exam 12d, 13
ENZYMES
Lehninger IV p. 190- 213
Active site. cofactor, coenzyme, prosthetic group; transition state (TS) and transition state stabilization. We reviewed transition state (TS) and transition state stabilization (example was urea hydrolysis and urease). We discussed the preferential stabilization of the TS.
Michaelis-Menten equation was covered with the underlying assumptions. Meanings of kcat and Km and catalytic efficiency. The concept of diffusion-controlled reactions was mentioned and ways to test this. Variation of rate as a function of enzyme concentration (enzyme assays). Multiple substrates and the concepts of random, ordered or ping-pong kinetics. Inhibition reversible and irreversible. Kinetics of competitive inhibitors and Lineweaver-Burke analysis. Strategies for designing a reversible inhibitor for a viral protease. Examples of irreversible inhibitors – diisopropylfluorophosphate and other fluorophosphates (targets and kinetics). Affinity labels and mechanism-based inactivators – concepts and kinetics.
pH effects on enzyme rates. Finally we will consider selected enzymes (including those showing allostery) when they arise in metabolism. ))
Problems for the enzyme section:
2006 2nd exam: 1, 2a, b
2006 Final: 9b, e, 10
2004 2nd exam: 1a, 1b, 1e, 1f, 3c, 5, 7
2004 Final: 6, 8d, 10
2002 2nd exam: 1a, 1b, 1c, 3, 4, 5, 8e, 10, 11,
2002 Final: 12f, 14b, 14c,
END OF EXAM 2 MATERIAL
CARBOHYDRATES
Carbohydrates Core Reading: Lehninger IV p. 238-253
Aldo- and keto- triose to hexoses. Numbering of open chains, D- vs. L-, epimers.
>>READINGS what’s next ….
Lipids and Membranes: Lehninger IV Chapter 10 (can omit the following pp. 352-354 and 363-367; skim pp. 357-363). Chapter 11 (but not pp. 383-389 and 393-416).
Bioenergetics and Metabolism Lehninger 481-488
Phosphoryl transfers Lehninger 496-502
Glycolysis (to start with) Lehninger 521-536