Proteins? Protein function. Protein folding. Protein folding diseases. Protein interactions. Macromolecular assemblies. The end product of Genes
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1 Proteins? Protein function Protein folding Protein folding diseases Protein interactions Macromolecular assemblies The end product of Genes
2 Protein Unfolding
3 DOD Acid Catalysis DOD HDOD + N H N D C N C C C N C C D + O R O C O R O C
4 OD - N H C R O C C O N C DOD N D C R O C C O N C HOD N H C R O C C O N C DOD D + DOD N D C R O C C O N C HDOD + Acid Catalysis Base Catalysis
5 Amide Proton Exchange is ph dependent 4 Log[k(sec-1)] ph
6 k op k ch Folded Open hydrogen exchange k cl N H N D EX 1 mechanism: k ch > k cl k ex = k op EX 2 mechanism: k ch < k cl k ex = K (op/cl) k ch ph dependent
7 Cooperative Unfolding Cytochrome C Englander, et. al. 2002
8 Cooperative Unfolding Cytochrome C Englander, et. al. 2002
9 Hoang, et. al Cytochrome C Folding Pathway
10 Multi-state vs Two State Protein Unfolding Englander, et. al. 2002
11 NMR H/D exchange Individual amide proton exchange rates Sensitive to subtle protein dynamics Used extensively to study protein folding Problems Need pure sample Need high concentrations Only small proteins
12 Mass Spec H/D exchange? David Smith, (Zhang et al 1993) Sample need not be pure Low sample concentrations Large proteins Macromolecular complexes Problems Digestion coverage Exchange rate is averaged over the whole peptide Buffer intolerance
13 H/D Exchange Experimental Protocol Protein Pepsin digestion (Optional) Exchange with D 2 0 Quench with low ph over time Fragment size residues 4 Log[k(sec-1)] ph Mass spectrometry Assignment of peptides Quantifying exchange
14 Protein stability pulsed labeling H/D exchange [GdmCL] Percentage exchange [GdmCl]
15 MALDI Analysis of Pulse labeled proteins SUPREX SUPREX; Stability of Unpurified Proteins from Rates of H/D Exchange Unpurified proteins Rapid analysis High throughput Protein stability in the cell Ghaemmaghami, et. al. 2000
16 SUPREX Suprex = CD = 4-oxalocrotonate tautomerase Powell, et. al. 2000
17 SUPREX analysis of mutations and protein stability Protein stability Maltose binding protein Ghaemmaghami, et. al. 2000
18 Association detected by SUPREX 10 mm = 56 mm = Trp repressor (TrpR) - L-tryptophan = + L-tryptophan = Powell, et. al. 2000
19 Ghaemmaghami, et. al Protein Stability in Cells
20 MALDI Analysis of Pulse labeled proteins SUPREX Unpurified proteins Rapid analysis High throughput Protein stability in the cell?
21 Continous H/D Exchange Protein folding Protein interfaces Quantitatively determine rates?
22 H/D Exchange Experimental Protocol CA Tubes or Dimers Pepsin digestion Exchange with D 2 0 Quench with low ph over time Fragment size residues Mass spectrometry Assignment of peptides Quantifying exchange
23 Identification of protein interaction interfaces Protein H H H H H H H H H H D 2 O D D D D D D D D D D Surface amide protons replaced with deuterons Surface amide protons H H H H H H D H D D H H H H Deuteriums trapped H H H H H H H 2 O D D D D D D D D D D Deuterium foot print
24 Detection of PKI Interaction with PKA Protonated Deuterated Off exchange (10 min) + ATP + ATP & PKI Mandell et al. 1998
25 PKI, PKA, and ATP PKI ATP PKA Mandell et al. 1998
26 mab epitope Epitope mapping of a monoclonal antibody against thrombin by H/D-exchange
27 Novel Approaches for Understanding Virus Assembly and Dynamics. Lanman et al. 2003
28 Image Reconstructions of Procapsid and Mature Virion
29 The Coat Protein Subunit has a Two Domain Structure HINGE N-terminal domain 21 kda C-terminal domain 25 kda Lanman, et. al. 1999
30 A domain-shuffling model for capsid expansion Unfolded Subunit Monomer Mutant Dimer N C Procapsid Expanded Lattice
31 The Model Predicts Trapping of Deuterium during Expansion Procapsid Transition State Expanded
32 Changes in Exchange Protection during Assembly & Maturation M PS ES X MALNEGQIVT LAVDEIIETI SAITPMAQKA KKYTPPAASM QRSSNTIWMP VEQESPTQEG 60 M PS ES X WDLTDKATGL LELNVAVNMG EPDNDFFQLR ADDLRDETAY RRRIQSAARK LANNVELKVA 120 M PS ES X NMAAEMGSLV ITSPDAIGTN TADAWNFVAD AEEIMFSREL NRDMGTSYFF NPQDYKKAGY 180 M PS ES X M PS ES X M PS ES X DLTKRDIFGR IPEEAYRDGT IQRQVAGFDD VLRSPKLPVL TKSTATGITV SGAQSFKPVA 240 M PS ES X WQLDNDGNKV NVDNRFATVT LSATTGMKRG DKISFAGVKF LGQMAKNVLA QDATFSVVRV 300 VDGTHVEITP KPVALDDVSL SPEQRAYANV NTSLADAMAV NILNVKDART NVFWADDAIR 360 M PS ES X IVSQPIPANH ELFAGMKTTS FSIPDVGLNG IFATQGDIST L SGLCRIALW YGVNATRPEA 420 monomer protection M PS ES X IGVGLPGQTA 430 procapsid shell protection expanded shell protection deuterium retention after expansion Tuma, et. al. 2000
33 HIV Assembly & Maturation Immature Mature TM SU MA CA RNA Gag NC Gag MA CA p2 NC p1 p6
34 Thin-Section EM Analysis of Assembly Products
35 CA is Comprised of Distinct N- and C- Terminal Structural Domains N-domain C-domain
36 CA Cylinders are Based on a Hexamer Lattice From Li et al, Nature 407:409 (2000)
37 Hypothesis: The CA that does not form dimers, because of a mutation at the dimer interface should form hexamers at high NaCl concentrations +NaCl
38 Hypothesis: The CA that does not form dimers, because of a mutation at the dimer interface should form hexamers at high NaCl concentrations +NaCl CA does not form hexamers with out C-domain (dimer) interaction.
39 H/D exchange analyzed with FT-MS
40 H/D Exchange Experimental Protocol CA Tubes or Dimers Pepsin digestion Exchange with D 2 0 Quench with low ph over time Fragment size residues Mass spectrometry Assignment of peptides Quantifying exchange
41 ESI-MS Analysis of Peptides Ice bath (4 C) Pump A H 2 O, 0.05% Formic acid Waste Sample loop Pump B ACN, 0.05% Formic acid C18 column MS Sample injection
42 Elution of Digest from C4 Column ~1 min Abundance Time
43 Representative FT-MS Scan m/z
44 Expanded View of m/z Region A = ( * 2) Da B B = ( * 2) Da C = ( * 1) Da A C m/z
45 With High Resolution Peptides of Similar m/z can be Analyzed within 1 ppm within 0.5 ppm
46 Peptides Covering 95% of the Sequence have Been Assigned 1 PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG 61 GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTH 121 NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE 181 VKNWMTETLL VQNANPDCKT ILKALGPGAT LEEMMTACQG VGGPGHKARV L
47 Expanded View of m/z Region A = ( * 2) Da B B = ( * 2) Da C = ( * 1) Da A C m/z
48 Extremely High Resolution is Achievable with FT-MS 40 mda m/z
49 The Related Peaks are Analyzed to Determine the Distribution m/z
50 Isotopic distributions during H/D exchange 0 min 0.5 min 2 h m/z
51 The Centroids of the Distribution are Calculated 0 min Centroid = min Centroid = h Centroid = m/z
52 Deuterium Incorporation at the Dimer Interface
53 Deuterium Incorporation Causes a Mass Increase H/D Exchange Period 0 sec
54 Deuterium Incorporation Causes a Mass Increase H/D Exchange Period 0 sec Assembled CA Unassembled CA 30 sec
55 Deuterium Incorporation Causes a Mass Increase H/D Exchange Period 0 sec Assembled CA Unassembled CA 30 sec 2 min 1 h 4 h 68 h m/z 849
56 The Dimer Interface is Protected Upon Assembly Centroid of Mass Time (min)
57 Helix II Shows Protection Upon Assembly 1272 Centroid of Mass Time (min)
58 Helices VI and VII Show Little Protection Centroid of Mass H1932mean M1932mean Data: Dmean1932mon_Mean Model: 3expHD Chi^2 = R^2 = A ± B ± C ± k ± k ± k ± Time (min)
59 The Bottom of Helix III becomes Protected Centroid of Mass Time (min)
60 The Dimer Interface is Protected Upon Assembly Centroid of Mass Time (min)
61 Helices VI and VII Show Little Protection Centroid of Mass H1932mean M1932mean Data: Dmean1932mon_Mean Model: 3expHD Chi^2 = R^2 = A ± B ± C ± k ± k ± k ± Time (min)
62 Changes in Exchange Rates due to CA Assembly Lanman, et. al. 2003
63 SEC separation of cross-linked CA monomer and dimer CA Monomer mau Absorbance at 280 nm CA Dimer CA Dimer CA Monomer ml Elution volume (ml)
64 Ion coun Mass spectra of the cross-linked species Time (min) Time (min) m/z m/z
65 Lysine 70 was cross-linked to Lysine to 131 = to 199 = DST = Expected mass = Lys 70 Observed mass = Lys70 cross-linked to 182 Lys 182
66 An N-domain to C-domain Interaction in CA Assembly B 1 2 A A 3 B
67 Three Sites of Interaction During Assembly N:N N:C C:C
68 Advantages of Hydrogen/Deuterium Exchange Small quantities required (10-12 mole) Needn t be pure No symmetry constraints Can provide time resolved or dynamic information
69 The future of H/D exchange Multi protein macromolecular complexes Cooperativity in large proteins or macromolecular complexes Exchange rates for individual amide protons Protein dynamics during motor motions
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