1. Protein Sequencing Primary Structure of Proteins
Chapter 3
Part 3

2. Learning Goals
Understand levels of protein structure. This part focuses on Primary Structure (Chapter 4 we will learn secondary, tertiary and quaternary structure).
Know N-terminal and C-terminal determination.
Principles of protein fragmentation.
Know the Edman degradation and the importance of overlapping sequences.
Electrospray Mass spectrometry and Tandem MS.
How protein sequences are used in understanding evolution of proteins and their functions (consensus sequences

3. Levels of Protein Structure
Review…what we will do here is determine primary sequence of a protein or peptide.

4. Fredrick Sanger ALSO developed one of the first successful DNA sequencing methods.
Fredrick Sanger – developed first N-terminal determination and early sequencing methods

5. Protein Sequencing
It is essential to further biochemical analysis that we know the sequence of the protein we are studying
Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage, and identification
Can be used to identify protein with known sequence
Mass Spectrometry (Modern method)
MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
Can be used to determine post-translational modifications.
Actual sequence can also be determined from DNA sequence

6. Insulin – the First Protein Sequenced
Once an pure protein is obtained, then….
What needs to be done ?
The first protein sequenced is tiny protein hormone. How many amino acids? Check out the positions of the disulfide bonds. Once insulin was purified…then what needs to be done? Another way to ask this question is: is each polypeptide pure?

7. R-S-S-R
Methods to reduce and keep disulfides from reforming. They should have put the formula for dithiothreitol on the other side of the slide.
For Insulin, then after doing this, then they had to separate EACH polypeptide to get each pure.

8. Acid Hydrolysis of Proteins
6 N HCl for several hours, oC,to hydrolyze Peptide bonds
Four Problems to Deal With:
1. Destroys W.
2. Partially (slowly) destroys S and T.
3. Converts N  D + NH4+ , and Q  E + NH4+.
4. Slowly hydrolyzes peptide bonds between vicinal ile, leu, and val.
Dealt with by:
1. KOH hydrolysis to determine W.
2. HCl hydrolysis over 2 hr, 4hr, 6hr….for S, T, I, L, V.
3. Measure NH4+ to determine amount of N+Q  D + E.
First things first…what aa’s does the polypeptide have? Both acid and basic hydrolysis are needed.

9. Acid Hydrolysis of Luciferase
S and T in the protein are extrapolated back to zero time before they could be degraded. I and V’s here were vicinal.

10. Amino Acid Analysis of AGDFRG
Amino acid analysis: 6N HCl hydrolysate run through an amino acid analyzer that uses ion-exchange columns with buffer changes to separate all of the amino acids. As the amino acids come off the column, they are mixed with ninhydrin (see next slide) to form a colored compound which can be measured in the flow through spectrophotometer. All the amino acids produce a purple color with the ninhydrin reaction except for proline which forms a yellow color.
Based on Ninhydrin Reaction

11. Amino Acid Analysis of Acid Hydrolysate
HPLC using ion exchange or other chromatography – all automated equipment.
Amino acids, as they come off the column, reacted with ninhydrin.
The important thing here is that ninhydrin is colorless, but the produce is intensely purple. You do not have to know this reaction, or ninhydrin’s structure for the exam. It is the analytical concept that is important.

12. N-terminal Reagents
N-terminal determination: the classic is Sanger’s F-dinitrobenzene (yellow), the other here are fluorescent. There are several reagents that can react with primary amino groups. These products (DNP-labeled amino acids) are stable to acid hydrolysis. So after labeling with FDNP to form the DNP-aa at the N-terminal end, the product is then acid hydrolyzed and using thin layer chromatography identify the N-terminal aa. Besides the N-terminal amino acid being labeled is there another one that will be labeled? If so, consider it’s position in the peptide and consequent labeling.

13. C terminal Carboxypeptidases

14. Hydrazinolysis
Polypeptide + anhyd-Hydrazine at 90oC + mildly acidic ion exchange resin (catalyst)  for hrs.
Hydrazinolysis modifies the peptide bond’s carboxyl to form amino acyl hydrazides. The only amino acid in the polypeptide to not form an amino acyl hydrazide is the C-terminal amino acid: a chemical method of C-terminal determination.

15. Edman Degradation
FIGURE 3–27 The protein sequencing chemistry devised by Pehr Edman. The peptide bond nearest to the amino terminus of the protein or polypeptide is cleaved in two steps. The two steps are carried out under very different reaction conditions (basic conditions in step 1, acidic in
step 2), allowing one step to proceed to completion before the second is initiated.

16. Edman Degradation = Amino Acid Sequencing
This is the reaction. See that after formation of the PTH-aa, what was amino acid 2 of the original peptide, is now N-terminal for the next round.
The PTH-aa’s (they only vary in the R-group) are identified automated equipment

17. Protein Fragmentation Methods
Edman degredation works on short peptides <20-30 aa’s. To do a whole protein from 100’s to 1,000’s aa long, they must be SPECIFICALLY fragmented followed by purification of each fragment, then the fragments can be sequenced by the Edman degredation. You need to know where the endopepditases trypsin, chymotrypsin, pepsin and the reagent cyanogen bromide cut: amino acid specificity and which side of the aa (each aa in a peptide is connected through 2 peptide bonds).
You need to know 4 of them: trypsin, chymotrypsin, pepsin and cyanogen bromide fragmentation methods.

18. Trypsin Fragmentation
Note that where trypsin cuts, it produces fragments whose C-terminal aa is characteristic of trypsin’s specificity. Other endopeptidases would make cuts at different specific sites.

19. CNBr Fragmentation Cleaves the C-terminal side of Met…
….and converts the Met to Homoserine Lactone
CNBr only cuts a M. And, during the reaction converts M to homoserine lactone. So that when the Edman degradation is done, it produces the PTH-homoserine lactone, which means that M was at that position.

20. Protein Sequencing Overall Flow
Each tryptic peptide has to be isolated pure before Edman degradation sequencing.

21. Trypsin and CNBr Fragmentation Each Done Separately
Comparison of two fragmentation methods.

22. Separation of Protein Fragments The Classic Paper Chromatography + Electrophoresis
In the beginning fragments were separated on 2-D paper electrophoresis-chromatography. Today HPLC is used. But the essential point is that each peptide fragment has different sizes, charge, solubility from the others so they can be cleanly separated so that the Edman degradation can be used to get the aa sequence of the peptide.

23. Protein Sequencing: Overlapping Sequences
The overview with the conclusions that can be drawn for each step. Note that in order to do this, two different fragmentation methods are needed so that the order of the fragments can be arranged to get the sequence of the total protein (using overlapping sequences to align the fragments).

24. Protein Sequence from DNA Sequence
We will see in Chapter 8 how to do DNA sequencing. Then finding the beginning of a gene, you can apply the universal genetic code to get the translation product of the gene which is the amino acid sequence. As technology has advanced, it turns out that DNA sequencing can easily do over 1000 nucleotides from a DNA fragment and these have been successfully automated so that high-throughput labs can sequence enormous lengths of DNA in a day.

25. MS Procedures for Sequence IDs
FIGURE 3–30 Electrospray ionization mass spectrometry of a protein. (a) A protein solution is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field. The droplets evaporate, and the ions (with added protons in this case) enter the mass spectrometer for m/z measurement. The spectrum generated (b) is a family of peaks, with each successive peak (from right to left) corresponding to a charged species increased by 1 in both mass and charge. The inset shows a computer-generated transformation of this spectrum.
FIGURE 3–31 Obtaining protein sequence information with tandem MS. (a) After proteolytic hydrolysis, a protein solution is injected into a
mass spectrometer (MS-1). The different peptides are sorted so that only one type is selected for further analysis. The selected peptide is further fragmented in a chamber between the two mass spectrometers, and m/z for each fragment is measured in the second mass spectrometer (MS-
2). Many of the ions generated during this second fragmentation result from breakage of the peptide bond, as shown. These are called b-type or
y-type ions, depending on whether the charge is retained on the amino- or carboxyl-terminal side, respectively. (b) A typical spectrum with peaks
representing the peptide fragments generated from a sample of one small peptide (21 residues). The labeled peaks are y-type ions derived from
amino acid residues. The number in parentheses over each peak is the molecular weight of the amino acid ion. The successive peaks differ by
the mass of a particular amino acid in the original peptide. The deduced sequence is shown at the top.

26. Chemical Synthesis of Polypeptides
You do not have to know the exact chemistry of this synthesis, but you do have to know it exists and allows biochemists to construct artificial peptides. The technology has, of course, been automated so that it is realativly simple to get any peptide made to order.

27. Consensus Sequences
Now…we compare similar proteins from different species and can discover consensus sequences. So for the top panel, the consensus sequence is:
G(A) __ __ __ __ G K T(S). The blanks can be any amino acid. These are made from alignment programs that aligns the primary sequence of one protein with another…see next slide. A

28. Partial Primary Structure of Elongation Factor Tu Sequences Aligned with Gaps
To align these sequences the alignment program had to insert a gap.

29. EF-Tu Signature Sequences
Signature sequences identify sequences shared universally among large taxonomic groups.

30. GroEL Phylogeny
Phylogenetic programs allow the construction of trees. Here the EF-Tu tree with the length of the branch being related to the number of mutational changes from the branch point. All from primary structure.

31. Cytochrome C
Cytochrome C sequence diversity was used to construct the cyt-c tree, next slide.

32. Cytochrome C Phyolgeny
This is expanded region of a larger tree and does not show other domains.

33. Proteins Evolve at Different Rates

34. Simple Sequencing Problem: What Each Part Tells You
Sequence of Leucine Enkephalin, a brain opioid peptide.
Complete hydrolysis by 6M HCl at 110oC followed by amino acid analysis indicated the presence of G, L, F, and Y in a 2:1:1:1 molar ratio.
This means the peptide could be 2:1:1:1, or 4:2:2:2, or….
Here is the problem…with data. From each data point you should be able to make conclusions about the peptide.

35. Simple Sequencing Problem: What Each Part Tells You
Sequence of Leucine Enkephalin, a brain opioid peptide.
Complete hydrolysis by 6M HCl at 110oC followed by amino acid analysis indicated the presence of G, L, F, and Y in a 2:1:1:1 molar ratio.
Treatment of the peptide with 1-fluoro-2,4, dinitrobenzene followed by complete hydrolysis and chromatography indicated the presence of 2,4-dinitrophenyl derivative of tyrosine. No free tyrosine could be found.
What does this tell you?
Anything about where in the peptide Y is?

36. Simple Sequencing Problem: What Each Part Tells You
Sequence of Leucine Enkephalin, a brain opioid peptide.
Complete hydrolysis by 6M HCl at 110oC followed by amino acid analysis indicated the presence of G, L, F, and Y in a 2:1:1:1 molar ratio.
Treatment of the peptide with 1-fluoro-2,4, dinitrobenzene followed by complete hydrolysis and chromatography indicated the presence of 2,4-dinitrophenyl derivative of tyrosine. No free tyrosine could be found.
What does this tell you?
Y is the N-terminal amino acid: so the peptide is Y _ _ _ _
Here is the problem…with data. From each data point you should be able to make conclusions about the peptide.

37. Simple Sequencing Problem: What Each Part Tells You
Sequence of Leucine Enkephalin, a brain opioid peptide.
Complete hydrolysis by 6M HCl at 110oC followed by amino acid analysis indicated the presence of G, L, F, and Y in a 2:1:1:1 molar ratio.
Treatment of the peptide with 1-fluoro-2,4, dinitrobenzene followed by complete hydrolysis and chromatography indicated the presence of 2,4-dinitrophenyl derivative of tyrosine. No free tyrosine could be found.
the peptide is Y _ _ _ _
c. Complete digestion of the peptide with chymotrypsin followed by chromatography yielded free tyrosine and leucine with a tripeptide containing Phe and Gly in a 1:2 ratio.
If you know where chymotrypsin fragments the peptide, it can only be one sequence. Try it before going to the next slide.

38. Simple Sequencing Problem: What Each Part Tells You
Sequence of Leucine Enkephalin, a brain opioid peptide.
Complete hydrolysis by 6M HCl at 110oC followed by amino acid analysis indicated the presence of G, L, F, and Y in a 2:1:1:1 molar ratio.
Treatment of the peptide with 1-fluoro-2,4, dinitrobenzene followed by complete hydrolysis and chromatography indicated the presence of 2,4-dinitrophenyl derivative of tyrosine. No free tyrosine could be found.
the peptide is Y _ _ _ _
Complete digestion of the peptide with chymotrypsin followed by chromatography yielded free tyrosine and leucine with a tripeptide containing Phe and Gly in a 1:2 ratio.
so… it is YGGFL
Here is the problem…with data. From each data point you should be able to make conclusions about the peptide.

39. Things to Know and Do Before Class
How to determine amino acid composition of a protein.
N-terminal determination, C-terminal determination.
Edman degradation: interpret results.
Protein Fragmentation methods + Overlapping Seqs.
Electrospray Mass Spectrometry (single and tandem) analysis of proteins.
Evaluation of protein evolution.
Do EOC problems 18, 19, 21, 22