Proteins? Protein function Protein folding Protein folding diseases Protein interactions Macromolecular assemblies The end product of Genes
Protein Unfolding
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
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
Amide Proton Exchange is ph dependent 4 Log[k(sec-1)] 2 0-2 -4 0 2 4 6 8 ph
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
Cooperative Unfolding Cytochrome C Englander, et. al. 2002
Cooperative Unfolding Cytochrome C Englander, et. al. 2002
Hoang, et. al. 2002 Cytochrome C Folding Pathway
Multi-state vs Two State Protein Unfolding Englander, et. al. 2002
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
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
H/D Exchange Experimental Protocol Protein Pepsin digestion (Optional) Exchange with D 2 0 Quench with low ph over time Fragment size 10-20 residues 4 Log[k(sec-1)] 2 0-2 -4 0 2 4 6 8 ph Mass spectrometry Assignment of peptides Quantifying exchange
Protein stability pulsed labeling H/D exchange [GdmCL] Percentage exchange 100 80 60 40 20 0 0 1 2 3 4 [GdmCl]
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
SUPREX Suprex = CD = 4-oxalocrotonate tautomerase Powell, et. al. 2000
SUPREX analysis of mutations and protein stability Protein stability Maltose binding protein Ghaemmaghami, et. al. 2000
Association detected by SUPREX 10 mm = 56 mm = Trp repressor (TrpR) - L-tryptophan = + L-tryptophan = Powell, et. al. 2000
Ghaemmaghami, et. al. 2001 Protein Stability in Cells
MALDI Analysis of Pulse labeled proteins SUPREX Unpurified proteins Rapid analysis High throughput Protein stability in the cell?
Continous H/D Exchange Protein folding Protein interfaces Quantitatively determine rates?
H/D Exchange Experimental Protocol CA Tubes or Dimers Pepsin digestion Exchange with D 2 0 Quench with low ph over time Fragment size 10-20 residues Mass spectrometry Assignment of peptides Quantifying exchange
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
Detection of PKI Interaction with PKA Protonated Deuterated Off exchange (10 min) + ATP + ATP & PKI Mandell et al. 1998
PKI, PKA, and ATP PKI ATP PKA Mandell et al. 1998
mab epitope Epitope mapping of a monoclonal antibody against thrombin by H/D-exchange
Novel Approaches for Understanding Virus Assembly and Dynamics. Lanman et al. 2003
Image Reconstructions of Procapsid and Mature Virion
The Coat Protein Subunit has a Two Domain Structure HINGE 175 204 N-terminal domain 21 kda C-terminal domain 25 kda Lanman, et. al. 1999
A domain-shuffling model for capsid expansion Unfolded Subunit Monomer Mutant Dimer N C 1 2 1 2 1 5 4 5 1 2 4 3 2 3 Procapsid Expanded Lattice
The Model Predicts Trapping of Deuterium during Expansion Procapsid Transition State Expanded
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
HIV Assembly & Maturation Immature Mature TM SU MA CA RNA Gag NC Gag MA CA p2 NC p1 p6
Thin-Section EM Analysis of Assembly Products
CA is Comprised of Distinct N- and C- Terminal Structural Domains N-domain C-domain
CA Cylinders are Based on a Hexamer Lattice From Li et al, Nature 407:409 (2000)
Hypothesis: The CA that does not form dimers, because of a mutation at the dimer interface should form hexamers at high NaCl concentrations +NaCl
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.
H/D exchange analyzed with FT-MS
H/D Exchange Experimental Protocol CA Tubes or Dimers Pepsin digestion Exchange with D 2 0 Quench with low ph over time Fragment size 10-20 residues Mass spectrometry Assignment of peptides Quantifying exchange
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 2 3 1 4 6 5 C18 column MS Sample injection
Elution of Digest from C4 Column ~1 min Abundance Time
Representative FT-MS Scan 600 800 1 000 m/z 1 200 1 400
Expanded View of 744-752 m/z Region A = (744.842 * 2) Da B B = (746.390 * 2) Da C = (749.386 * 1) Da A C 745 746 747 748 m/z 749 750 751 752
With High Resolution Peptides of Similar m/z can be Analyzed 744.842 within 1 ppm 746.390 within 0.5 ppm
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
Expanded View of 744-752 m/z Region A = (744.842 * 2) Da B B = (746.390 * 2) Da C = (749.386 * 1) Da A C 745 746 747 748 m/z 749 750 751 752
Extremely High Resolution is Achievable with FT-MS 40 mda 745 746 747 748 m/z 749 750 751 752
The Related Peaks are Analyzed to Determine the Distribution 745 746 747 748 m/z 749 750 751 752
Isotopic distributions during H/D exchange 0 min 0.5 min 2 h 746 748 750 m/z
The Centroids of the Distribution are Calculated 0 min Centroid = 745.5 0.5 min Centroid = 748 2 h Centroid = 749 746 748 750 m/z
Deuterium Incorporation at the Dimer Interface
Deuterium Incorporation Causes a Mass Increase H/D Exchange Period 0 sec
Deuterium Incorporation Causes a Mass Increase H/D Exchange Period 0 sec Assembled CA Unassembled CA 30 sec
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 843 846 m/z 849
The Dimer Interface is Protected Upon Assembly 2542 2540 Centroid of Mass 2538 2536 2534 2532 2530 1 10 100 1000 Time (min)
Helix II Shows Protection Upon Assembly 1272 Centroid of Mass 1271 1270 1269 1268 1267 1 10 100 1000 Time (min)
Helices VI and VII Show Little Protection Centroid of Mass 1940 1939 1938 1937 1936 1935 H1932mean M1932mean Data: Dmean1932mon_Mean Model: 3expHD Chi^2 = 0.04466 R^2 = 0.99146 A 1.45506 ±0.42438 B 1.619 ±0.41511 C 5.59556 ±0.32398 k1 0.08275 ±0.04485 k2 0.00548 ±0.00358 k3 0.00015 ±0.00002 1934 1933 1 10 100 1000 Time (min)
The Bottom of Helix III becomes Protected 1498 1497 Centroid of Mass 1496 1495 1494 1493 1492 1491 1490 1 10 100 1000 Time (min)
The Dimer Interface is Protected Upon Assembly 2542 2540 Centroid of Mass 2538 2536 2534 2532 2530 1 10 100 1000 Time (min)
Helices VI and VII Show Little Protection Centroid of Mass 1940 1939 1938 1937 1936 1935 H1932mean M1932mean Data: Dmean1932mon_Mean Model: 3expHD Chi^2 = 0.04466 R^2 = 0.99146 A 1.45506 ±0.42438 B 1.619 ±0.41511 C 5.59556 ±0.32398 k1 0.08275 ±0.04485 k2 0.00548 ±0.00358 k3 0.00015 ±0.00002 1934 1933 1 10 100 1000 Time (min)
Changes in Exchange Rates due to CA Assembly Lanman, et. al. 2003
SEC separation of cross-linked CA monomer and dimer CA Monomer mau Absorbance at 280 nm 400 300 200 100 CA Dimer CA Dimer CA Monomer 0 0 20 40 60 80 100 ml 0 20 40 60 Elution volume (ml) 80 100
Ion coun Mass spectra of the cross-linked species Time (min) Time (min) 11+ 10+ 14439 12+ 9+ 13+ 8+ 1000 1500 2000 10000 15000 20000 m/z m/z
Lysine 70 was cross-linked to Lysine 182 31 to 131 = 10948.9 171 to 199 = 3375.9 +DST = 114.0 Expected mass = 14438.8 Lys 70 Observed mass = 14439 Lys70 cross-linked to 182 Lys 182
An N-domain to C-domain Interaction in CA Assembly B 1 2 A A 3 B
Three Sites of Interaction During Assembly N:N N:C C:C
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
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