1. Protein structure

2. Secondary structure α-helix β-sheet: parallel and anti-parallel β-turn
H-bonds between N-H and C=O in peptide backbone

3. Protein Quarternary Structure

4. a2 The Cro protein of bacteriophage l is a dimer “homodimer”
An example of quaternary structure

5. Hemoglobin is a tetramer
a2b2

6. What determines three dimensional structure?
Answer: Amino acid sequence How do we know this?

7. Bovine ribonuclease primary structure
secreted pancreatic protein that degrades RNA
very stable
early “model” protein

8. Two denaturants and a reductant

9. Reduction of protein disulfides by b-mercaptoethanol

10. Complete denaturation of ribonuclease by urea and b-mercaptoethanol
Enzymatically active
Enzymatically inactive

11. Spontaneous renaturation by removal of urea and b-mercaptoethanol+ -
sequence contains all the “information” needed to specify the correct structure of ribonuclease
this experiment does not work with all proteins, however

12. When reoxidized in the presence of denaturant, disulfides are scrambled
Enzymatically inactive
driven by the decrease in free energy (-DG) as the scrambled conformations (less stable) are converted to native conformation (most stable)
Enzymatically active

13. One amino acid sequence – one 3D structure?
Most of the time
Exceptions:
Intrinsically unstructured proteins
Metamorphic proteins

14. Lymphotactin, a metamorphic protein

15. Protein misfolding: amyloid form of human prion protein
Creutzfeld–Jacob disease (CJD) in humans; BSE (mad cow disease) in cows

16. Transmission of prion protein diseases
The infective agent is a misfolded protein

17. Some protein side chains are covalently modified

18. Different ways of representing protein structure
Will use protein structure visualization software in Tutorial 2

24. Determining amino acid
sequences

28. Sequence comparisons

29. Evolutionary relationships

30. Protein structure relationships
Same function
Different function!

31. Homologous proteins are derived from a common ancestor
between species
same or very similar functions
within species
different functions

32. Sequence comparisons: what can we learn?

33. How do we find the “best” alignment?
Simplest approach: “no frills”

34. Gap insertion in alignment
now, there are 38 identities
a “penalty” is paid for each gap that is introduced

35. Scoring alignments
scoring system: each identity between aligned sequences is counted as +10 points, whereas each gap introduced, regardless of size, counts for −25 points.

36. Shuffling is used to test for randomness of the alignment

37. Actual alignment score is compared with the scores for shuffled sequences

38. Substitution matrices are used to score for similar amino acids

39. How the matrix is used to assign a score for a substitution

40. Alignment with gap: identities and conservative substitutions

41. Myglobin vs. leghemoglobin alignments: comparison of different methods
red = alignments using authentic sequences
Accounting for aa similarity leads to better separation of authentic vs shuffled sequences

42. Myglobin vs. leghemoglobin alignment using the Blosum-62 matrix
23% identity
For proteins >100 aa:
sequence identities > 25% unlikely due to chance alone and are likely homologs
< 15% identical, alignment alone is unlikely to indicate statistically significant similarity
between 15 and 25% identical, further analysis needed to determine the statistical significance

43. BLAST (Basic Local Alignment Search Tool) search output