1. Protein structure depends on amino acid sequence and interactions Amino acid sequence Local interactions Long distance interactions Interactions between subunits

3. Other methods?

6. Tools for protein topology studies Identification of the amide bond cleaved by a single proteolytic event that leads to complementary peptides Complementary proteolysis Identification of the individual residue modified by, even aspecific mono-bifunctional, reagents Selective chemical modification Hydrogen/deuterium exchange Quantitative analysis of amide bonds accessible to solvent and available to exchange

7. Sequence/Structure Paradigm higher order structures are the main determinants of the preferential cleavage site. peptide bonds buried in the protein core and/or located within rigid secondary structures are less accessible to proteases

8. Proteolytic cleavage site HPLC ESMS ARG 93 COMPLEMENTARY PROTEOLYSIS

9.  -lactalbumin

10. RP-HPLC Conformational changes A D min Abs Protein under different experimental conditions (ligands, denaturants, pH etc…) A D Proteolysis under controlled conditions Native protein A C A C Proteolysis under controlled conditions min Abs AC B Exposed and flexible sites B

11. LC- MS LC- MS/MS

12. Proteolysis Experiment Conditions Proteases –Trypsin, Lys-C, Chymotrypsin, GluC, ………others Buffer condition –pH, salt and detergent if necessary--Protein native condition? Protein concentration Time points –5min, 10min, 30min, 1h, 2hrs and 4hrs.

13. Complementary proteolysis

14. H/D exchange and mass spectrometry Very slow Very fast N H CHC O N H CHC ORR Strongly depending on structural environment

17. H/D-MS measures the rate at which peptide amide hydrogens exchange with the hydrogen in the water where the protein is dissolved. The rate of exchange reveals the degree of exposure of each amide hydrogen in the folded protein to water. In the unfolded portions, this rate is higher than that of structured, folded sections. Factors that Slow Exchange Rate  Hydrogen bonding that creates the secondary structure, primarily alpha-helices and beta sheets.  Protection from the solvent, primarily due to being buried in the hydrophobic core of the protein.  Hydrogen bonding to water in the solvent (much smaller effect than the previous two).

18. HPLC ESI Probe Before After Protein D2OD2O Labelled protein Acid quenching H/D exchange esperiments by ESIMS Chung E.W. et al. (1997) Protein Science : 6, 1316 - 24

19. HPLC ESI Probe Labelled peptides Before After Pepsin digestion 5min, pH 2, 0°C Labelled protein Wang L. et al. (2002) Mol. Cell.Proteomics: 1, 132-138

20. HOLO - Ca 2+ APO HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) M. Svensson et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4221- 4226 - Ca 2+ HAMLET Oleic acid

21. Intensity (%) m /z HAMLETapoholo Global H/D exchange profile 1 min 5 min 60 min 15 min m/z 15 sec 0% 1759.77 0% 1759.77 0% 1759.77 100% 1777,27 100% 1777,27 100% 1777,27

22. 20 140 0 60 Time (min) H/D exchanged holo ~80 HAMLET ~130 apo* apo ~110 Hydrogen -Deuterium Exchange

23. 1 KQFTKCELSQ LLKDIDGYGG IALPELICTM FHTSGYDTQA IVENNESTEY GLFQISNKLW 60 61 CKSSQVPQSR NICDISCDKF LDDDITDDIM CAKKILDIKG IDYWLAHKAL CTEKLEQWLC 120 121 EKL 123 A B 3 10 C 3 3 D 1 2 3 4 5 6 3 1 Pepsin digestion for the local exchange profile

24. No substantial difference between HAMLET and Apo in peptides from the α domain Holo α-lactalbumin is always less flexible than HAMLET and Apo HAMLET incorporates a higher percentage of deuterium than Apo in peptides from the β domain HAMLET Apo α-actalbumin Holo α-lactalbumin 2 (12-23)

25. Asp37 Tyr50 Phe53 Gln39 Arg70 Glu49 Glu46 HAMLET/apo differential topology

26. Chemical crosslinking

32.  Hsp26 is an ATP independent cytosolic chaperone of Saccharomyces cerevisiae. Saccharomyces cerevisiae Hsp26  Hsp26 expression is related to environmental stress (i.e. temperature).  Hsp26 has the ability to form Dimers and large Oligomers of 24 subunits.  Hsp26 acts as a buffer for (partly) unfolded proteins, handing them over to other chaperon-systems, such as Hsp70.

33. N substrateaggregate I ΔTΔT Hsp26 oligomer ΔTΔT Hsp26 dissociated Hsp26-substrate complex N Disassembling/Assembling of the oligomer plays a vital role in chaperon function

34. Chemical crosslinking C O HO NH 2 LysGlu, Asp EDAC C O N H Legame isopeptidico CH 3 CH 2 NCN (CH 2 ) 3 HCl - CH 3 N+N+ EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

35. in situ digestion MALDI-TOF analysis Identification of crosslinked peptides

36. globular domainα-crystalline domainC-term. tailN-term. trimer domainglobular domainα-crystalline domainC-term. tailN-term. trimer domain 116 KDIDIEYHQNK 126 … 147 VKVKESSSGK 156 190 LKPQK 194 … 198 NHVKK 20 2 25°C43°C 151 ESSSGKFK 158 23 LLGEGGLRGYAPR 35 Chemical crosslinking