I.
Quizzes will be returned (temporarily) at the end of discussion today
Answers to handout questions will be posted the following week
II.
Objectives: Understand principle of spontaneous assembly. Know what interactions maintain each order of structure
Spontaneous assembly
Depictions: Ribbon and space-filled models of actin
Classes: Fibrous or globular ex. actin with myosin
III.
Objectives: Be able to make secondary structure predictions. Know how to use a helix wheel.
Analyzing amino acid sequences
1) Note sidechain personalities
2) Look for beta-bends. Pro, gly/ala, favorable interactions
3) Look for alpha-helices. No helix breakers, at least 20 aa
4) Look for patterns. Helix wheel may help
Questions:
1) Which secondary structures can be predicted from the primary sequence?
2) Add another strand to the beta-stands below, in correct configuration:
Antiparallel Parallel
3) In a beta-pleated sheet, how are the sidechains oriented in three-dimensional space?
4) Which is stronger, an antiparallel or parallel beta-sheet? Why?
5) Which is stronger, a beta-sheet (from #4) or an alpha-helix? Why?
6) From problems book Peptides and Proteins #14:
Would each of the following polypeptides be more likely to form an alpha-helix or a beta-pleated sheet in an aqueous environment at physiological pH?
ile-thr-leu-asp-phe-ser-met-glu-val-his-ala-pro-ala-ser-thr-val-arg-cys-gln-phe-lys-ile-asn-trp
ile-his-arg-cys-val-ser-asp-leu-ala-asn-ser-val-phe-ser-glu-met-ala-lys-ala-leu-cys-glu
IV.
Objectives: Be able to tackle these types of problems effortlessly and correctly!
Steps in primary structure determination:
1) Acid hydrolysis to get aa composition and length of peptide. Set spaces.
2) N-terminus (Sangers, Dansyl chloride, Edman) and C-terminus identification (reduction, hydrazinolysis, carboxypeptidases A, D, C, Y)
3) Endopeptidases and chemical reagents are your arsenal for sequencing
C-terminal proteases cut after a specific aa---trypsin, chymotrypsin, elastase, endo V8
N-terminal proteases cut before a specifc aa---themolysin, pepsin
And CnBr (like a C-terminal protease)
4) Map with overlapping fragments
Example:
Acid hydrolysis resulted in the following aa composition:
Ala, arg, glu, gly, leu, lys2, phe, trp, val
Treatment with Carboxypeptidase A yielded lys as the predominant species
One cycle of Edman degradation yielded a PTH-glu derivative
Enzymatic digestion with chymotrypsin yielded 3 fragments of the following aa compositions:
glu, phe
ala, arg, leu, lys, trp, val
gly, lys
Enzymatic digestion with trypsin yielded 3 fragments of the following aa compositions:
glu, lys, phe, val
ala, arg, leu
gly, lys, trp
Using these experimental observations, indicate possible sequence(s) of this peptide.
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