Time (min)
Undergraduate Category: Physical and Life Sciences Degree Level: B.S. Chemistry Abstract ID# 542
Dynamics of the SH3 Domains of Tec Family Tyrosine Kinases by Hydrogen Exchange Mass Spectrometry Justin M. Roberts, Thomas E. Wales, and John R. Engen Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
ABSTRACT
RESULTS AND DISCUSSION Itk SH3
Raw Mass Spectra +6
= EX1 unfolding
Time in D2O
Btk SH3 Tec SH3 +6
+6
UND
UND
10 s
UND
10 s
Raw Mass Spectra
+11
+6
UND
10 s
Txk SH3 Bmx SH3
10 s
10 s
3m
1m
3m
UND
1m
10 s
5m
BACKGROUND
7m
When the rate of deuterium incorporation is faster than the rate of refolding (k-1), we can observe EX1 kinetics.
Relative Deuterium Level (Da)
Peptide
Data
8976 9000
Mass
3 times in D2O 9024 shown
70
50
60
processing
Peptide MS Spectra
0 0.1
10
Time in Deuterium (min)
Deuterium incorporation graphs
423
425 m/z
427
3 times in D2O shown
40
50
80
40
60
20
20
20
10
10
10
10
0
0
0
0
1000
0.1
t1/2 = 5.34 min
1
10
100
1000
100
0.1
1
10
100
1000
+3
20
UND
0
0.1
1
10
100
1000
0.1
40
40
40
35
35
35
35
30
30
30
t1/2 = 6.67 min
25
25
25
25
25
20
20
20
20
20
15
15
15
15
15
10
10
10
10
10
5
5
5
5
5
0
0
0
0
0
1
10
100
1000
0.1
1
10
100
1000
0.1
1
10
100
1000
Electrospray MS
1
2
2.00
3
4
4.00
5
6
0.1
1
10
100
1000
1
10
100
5m
Tec
Itk Btk Tec Txk Bmx Lck Lyn Hck Fp1 Fyn Fgr Yes Src α-spec Abl Drk Amph2
Src
Sequence Alignment of Tec-family, Srcfamily, and 4 other SH3 domains
β1
RT loop
β2
1
10
100
n-Src loop
Distal loop
β4
310 helix
β5
171PEETLVIALYDYQTNDPQELALRCDEEYYLLDS-SEIHWWRVQD-KNGHE--GYAPSSYLVEKSP-231
Electrospray MS
2.00
4.00
6.00
Time (min)
10 10 Time (min)
β3
1000 1000
423
425 m/z
427
β4
β2
209-225
5
β1
44
N
3 22
n-Src loop
C
1
00 0.1 0.1
10 10 Time (min)
β5
1000 1000
310-helix
Btk SH3 233-242
Btk SH3
66
5
RT-loop
Distal loop
44 3
β4
22 1 00 0.1 0.1
10 10
1000 1000
233-242
Btk SH3 217-222 5
β3
β2
44
β1
3
N
C
22 1 00 0.1 0.1
10 10 Time (min)
n-Src loop
1000 1000
217-222
214SELKKVVALYDYMPMNANDLQLRKGDEYFILEE-SNLPWWRARD-KNGQE--GYIPSNYVTEAED-274 179NSEEIVVAMYDFQAAEGHDLRLERGQEYLILEK-NDVHWWRARD-KYGNE--GYIPSNYVTGKKS-239 82EEKIQVKALYDFLPREPCNLALRRAEEYLILEK-YNPHWWKARD-RLGNE--GLIPSNYVTENKI-142 206------LAQYD--SNSKKIYGSQPNFNMQYIPREDFPDWWQVRKLKSSSSSEDVASSNQKE-----269
CONCLUSIONS
61LQDNLVIALHSYEPSHDGDLGFEKGEQLRILE--QSGEWWKAQSLTTGQE--GFIPFNFVAKANS-121 63EQGDIVVALYPYDGIHPDDLSFKKGEKMKVLE--EHGEWWKAKSLLTKKE--GFIPSNYVAKLNT-123 78SEDIIVVALYDYEAIHHEDLSFQKGDQMVVLE--ESGEWWKARSLATRKE--GYIPSNYVARVDS-138 1----EFIAIYDYKAETEEDLTIKKGEKLEIIE--KEGDWWKAKAIGSGEI--GYIPANYIAAAE--56
82TGVTLFVALYDYEARTEDDLSFHKGEKFQILNS-SEGDWWEARSLTTGET--GYIPSNYVAPVDS-143 77IGVTLFIALYDYEARTGDDLTFTKGEKFHILNN-TEYDWWEARSLSSGHR--GYVPSNYVAPVDS-138
• Itk and Btk SH3 showed EX1 exchange kinetics with Itk demonstrating two distinct populations within the raw mass spectra.
91GGVTIFVALYDYEARTTEDLSFKKGERFQIINN-TEGDWWEARSIATGKS--GYIPSNYVVPADS-152 81GGVTTFVALYDYESRTETDLSFKKGERLQIVNN-TEGDWWLAHSLTTGQT--GYIPSNYVAPSDS-142
967TGKELVLALYDYQEKSPREVTMKKGDILTLLNS-TNKDWWKVEV--NDRQ--GFVPAAYVKKLDP-1026 61NDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQT--KNGQ--GWVPSNYITPVNS-121 1---MEAIAKHDFSATADDELSFRKTQILKILNMEDDSNWYRAEL--DGKE--GLIPSNYIEMKNYH58
• The reasons for these kinetics are not immediately evident from sequence homologies.
SRC Lyn (20m)
Hck (19m)
Fp1 (42s)
Fyn Fgr (5m) Yes
Lck (12s)
Src (5m) Abl1 (10m)
515GFMFKVQAQHDYTATDTDELQLKAGDVVLVIPF-QDEGWLMGVKESEKCR--GVFPENFTERVQ--588
6.00
3 times in D2O shown
1000
30 m
β3
190-197
66
10 m 0.1
RT-loop
Distal loop
Time (min)
Time (min)
MSE
1000
10 s
7
UPLC of pepsin fragments, 0°C
615
40
40
30
613 m/z
Btk SH3 233-242
3 times in D2O shown
peptide ID
1000
50
30
100
611
Max = 110 Da
60
30
10
m/z
4h
70
60
30
1
m/z Max = 70 Da
70
40
20
1216 1220 1224
Max = 73 Da
30
7m
1492 1496 1500
m/z
50
40
1496 1500 1504
4h
4h
Blue letters indicate residues involved in EX1 unfolding within each domain.
4 2
Max = 70 Da
4h
Itk SH3
Itk SH3 190-197
Desalting of protein, 0°C
20
6
deuterium
No digestion, 0°C, pH 2.5
Protein MS Spectra
0
25 m
Time (min)
Protein secondary structure is shown at the top.
hydrogen
Protein
Max = 59 Da
30
Pepsin digestion, 0°C, pH 2.5
40
m/z
35
Workflow4
60
m/z
40
Peak Width (Da)
4h 1476 1480 1484
60
0.1
Continuous labeling hydrogen exchange experiments were performed at room temperature. Labeled samples were injected into a Shimadzu HPLC (LC-20AD) coupled to a Waters Micromass LCT PremierXE Mass Spectrometer for global exchange. Online pepsin digestion was performed (Itk, Btk) and the peptides separated using a custom nanoACQUITY coupled to a Waters Qtof Premier Mass Spectrometer equipped with ESI source for peptide level analysis. Data were analyzed using MassLynxV4.1, PLGS 2.5 and DynamX 2.0 (Waters).
Backbone amides become deuterated
4h
1244 1248 1252 1256
Relative Deuterium Level (Da)
Recombinant Tec family SH3 domains (Tec, Txk, Itk, Btk, Bmx) were overexpressed in E. coli. Protein domains were purified by either nickel affinity and size exclusion chromatography (Tec, Txk, Btk, Bmx) or as a glutathione-S-transferase (GST)-tagged fusion protein (Itk).
Quench exchange reaction at various times, 0°C, pH 2.5
25 m
Relative Deuterium (Da)
4h
METHODS
D2O buffer
25 m
Relative Deuterium (Da)
EX1 Exchange Mechanism4
0.1
HXMS
10 m
5m
Because deuterium incorporation makes the protein heavier, it can be measured with a mass spectrometer. Uptake of deuterium is a function of backbone hydrogen bonding, solvent accessibility, and time—therefore we can uncover protein dynamics using HXMS. Usually, a protein gains mass gradually over time (EX2), but there are unique examples of cooperative unfolding (right), that results in a “jump” in mass. We investigate these EX1 events in the Tec family SH3 domains and related proteins.
Interpretation in light of structure, if known
5m
Relative Deuterium (Da)
Deuterium oxide—colloquially “heavy water”—contains two deuterium atoms, each heavier than the hydrogen in H2O by 1 neutron. Proteins are made up of amino acid chains held together by peptide (amide) bonds. By diluting protein in deuterium oxide (D2O), the backbone amide hydrogens can undergo exchange with deuterium:
Equilibrate 25°C, formulated pH
10 m
1m
10 m
same pH and temperature
3m
9 88 7 66 5 44 3 22 1 00 0.1 0.1
β1
β2-RT Loop
Itk SH3 209-225
Itk SH3 209-225 +3
3m
β3-Distal Loop-β4
EX1 kinetics in:
UND
Mapping of EX1 Regions onto Protein Structure
Deuterium Uptake Plots
Relative Deuterium (Da)
The Src Homology 3 (SH3) domain is an important regulatory domain found in many proteins.1 Using hydrogen exchange mass spectrometry (HXMS), the physical motions of domains such as this can be studied in solution.2 Previously, HXMS studies on the SH3 domains of the Src family tyrosine kinases showed that the native state dynamics of this domain varies on a wide time scale.3 Here we extend our studies to the SH3 domains of the Tec family kinases and characterize their unique dynamics using HXMS. We found that some Tec members, like their Src “cousins” undergo an uncommon slow kinetic reversible unfolding event—a structural phenomenon possibly related to function. We characterize this event (termed EX1 kinetics) and localize unfolding regions within the protein by cutting the protein up into peptides with an aspartic protease, and analyzing the pieces with mass spectrometry. Shifts in these unique dynamics may be further utilized to explore protein-protein interactions, such as HIV accessory protein binding and viral high-jacking of these immune-related regulatory domains.
REFERENCES [1] Zarrinpar, A., R. P. Bhattacharyya, et al. (2003). Sci. STKE 2003(179): re8-. [2] Wales, T. E.; J. R. Engen (2006). Mass Spectrometry Reviews 25(1): 158-170. [3] Wales, T. E.; J. R. Engen (2006. J. Mol. Biol. 357(5): 1592-1604. [4] Marcsisin, S.; J. Engen (2010). Anal. Bioanal. Chem. 397(3): 967-972.
ACKNOWLEDGEMENTS We would like to thank Sreya Tarafdar and the Smithgall lab for providing us with protein, and the Andreotti lab for providing us with the plasmid containing the Itk mouse SH3 domain. We are grateful to the NIH (GM101135), the Northeastern Provost grant, and the Honors Junior-Senior Project Grant for funding.
• The results of this experiment augment previous studies indicating that the SH3 domain is a dynamic domain in solution with varying temporal and spatial degrees of EX1 unfolding. The figure to the right illustrates SH3 kinetics for many SH3 domains, including EX1 half life, degree of EX1 kinetics, and domain phylogeny based on sequence alignment.
Drk (1.2s) Amph2 (1hr) α-Spec (16.5m)
Btk (6.8m) Itk (6.3m)
TEC
Tec Txk
Bmx
Dynamics of the SH3 Domains of Tec Family Tyrosine Kinases by Hydrogen Exchange Mass Spectrometry Justin M. Roberts, Thomas E. Wales, and John R. Engen Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
ABSTRACT
RESULTS AND DISCUSSION Itk SH3
Raw Mass Spectra +6
= EX1 unfolding
Time in D2O
Btk SH3 Tec SH3 +6
+6
UND
UND
10 s
UND
10 s
Raw Mass Spectra
+11
+6
UND
10 s
Txk SH3 Bmx SH3
10 s
10 s
3m
1m
3m
UND
1m
10 s
5m
BACKGROUND
7m
When the rate of deuterium incorporation is faster than the rate of refolding (k-1), we can observe EX1 kinetics.
Relative Deuterium Level (Da)
Peptide
Data
8976 9000
Mass
3 times in D2O 9024 shown
70
50
60
processing
Peptide MS Spectra
0 0.1
10
Time in Deuterium (min)
Deuterium incorporation graphs
423
425 m/z
427
3 times in D2O shown
40
50
80
40
60
20
20
20
10
10
10
10
0
0
0
0
1000
0.1
t1/2 = 5.34 min
1
10
100
1000
100
0.1
1
10
100
1000
+3
20
UND
0
0.1
1
10
100
1000
0.1
40
40
40
35
35
35
35
30
30
30
t1/2 = 6.67 min
25
25
25
25
25
20
20
20
20
20
15
15
15
15
15
10
10
10
10
10
5
5
5
5
5
0
0
0
0
0
1
10
100
1000
0.1
1
10
100
1000
0.1
1
10
100
1000
Electrospray MS
1
2
2.00
3
4
4.00
5
6
0.1
1
10
100
1000
1
10
100
5m
Tec
Itk Btk Tec Txk Bmx Lck Lyn Hck Fp1 Fyn Fgr Yes Src α-spec Abl Drk Amph2
Src
Sequence Alignment of Tec-family, Srcfamily, and 4 other SH3 domains
β1
RT loop
β2
1
10
100
n-Src loop
Distal loop
β4
310 helix
β5
171PEETLVIALYDYQTNDPQELALRCDEEYYLLDS-SEIHWWRVQD-KNGHE--GYAPSSYLVEKSP-231
Electrospray MS
2.00
4.00
6.00
Time (min)
10 10 Time (min)
β3
1000 1000
423
425 m/z
427
β4
β2
209-225
5
β1
44
N
3 22
n-Src loop
C
1
00 0.1 0.1
10 10 Time (min)
β5
1000 1000
310-helix
Btk SH3 233-242
Btk SH3
66
5
RT-loop
Distal loop
44 3
β4
22 1 00 0.1 0.1
10 10
1000 1000
233-242
Btk SH3 217-222 5
β3
β2
44
β1
3
N
C
22 1 00 0.1 0.1
10 10 Time (min)
n-Src loop
1000 1000
217-222
214SELKKVVALYDYMPMNANDLQLRKGDEYFILEE-SNLPWWRARD-KNGQE--GYIPSNYVTEAED-274 179NSEEIVVAMYDFQAAEGHDLRLERGQEYLILEK-NDVHWWRARD-KYGNE--GYIPSNYVTGKKS-239 82EEKIQVKALYDFLPREPCNLALRRAEEYLILEK-YNPHWWKARD-RLGNE--GLIPSNYVTENKI-142 206------LAQYD--SNSKKIYGSQPNFNMQYIPREDFPDWWQVRKLKSSSSSEDVASSNQKE-----269
CONCLUSIONS
61LQDNLVIALHSYEPSHDGDLGFEKGEQLRILE--QSGEWWKAQSLTTGQE--GFIPFNFVAKANS-121 63EQGDIVVALYPYDGIHPDDLSFKKGEKMKVLE--EHGEWWKAKSLLTKKE--GFIPSNYVAKLNT-123 78SEDIIVVALYDYEAIHHEDLSFQKGDQMVVLE--ESGEWWKARSLATRKE--GYIPSNYVARVDS-138 1----EFIAIYDYKAETEEDLTIKKGEKLEIIE--KEGDWWKAKAIGSGEI--GYIPANYIAAAE--56
82TGVTLFVALYDYEARTEDDLSFHKGEKFQILNS-SEGDWWEARSLTTGET--GYIPSNYVAPVDS-143 77IGVTLFIALYDYEARTGDDLTFTKGEKFHILNN-TEYDWWEARSLSSGHR--GYVPSNYVAPVDS-138
• Itk and Btk SH3 showed EX1 exchange kinetics with Itk demonstrating two distinct populations within the raw mass spectra.
91GGVTIFVALYDYEARTTEDLSFKKGERFQIINN-TEGDWWEARSIATGKS--GYIPSNYVVPADS-152 81GGVTTFVALYDYESRTETDLSFKKGERLQIVNN-TEGDWWLAHSLTTGQT--GYIPSNYVAPSDS-142
967TGKELVLALYDYQEKSPREVTMKKGDILTLLNS-TNKDWWKVEV--NDRQ--GFVPAAYVKKLDP-1026 61NDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQT--KNGQ--GWVPSNYITPVNS-121 1---MEAIAKHDFSATADDELSFRKTQILKILNMEDDSNWYRAEL--DGKE--GLIPSNYIEMKNYH58
• The reasons for these kinetics are not immediately evident from sequence homologies.
SRC Lyn (20m)
Hck (19m)
Fp1 (42s)
Fyn Fgr (5m) Yes
Lck (12s)
Src (5m) Abl1 (10m)
515GFMFKVQAQHDYTATDTDELQLKAGDVVLVIPF-QDEGWLMGVKESEKCR--GVFPENFTERVQ--588
6.00
3 times in D2O shown
1000
30 m
β3
190-197
66
10 m 0.1
RT-loop
Distal loop
Time (min)
Time (min)
MSE
1000
10 s
7
UPLC of pepsin fragments, 0°C
615
40
40
30
613 m/z
Btk SH3 233-242
3 times in D2O shown
peptide ID
1000
50
30
100
611
Max = 110 Da
60
30
10
m/z
4h
70
60
30
1
m/z Max = 70 Da
70
40
20
1216 1220 1224
Max = 73 Da
30
7m
1492 1496 1500
m/z
50
40
1496 1500 1504
4h
4h
Blue letters indicate residues involved in EX1 unfolding within each domain.
4 2
Max = 70 Da
4h
Itk SH3
Itk SH3 190-197
Desalting of protein, 0°C
20
6
deuterium
No digestion, 0°C, pH 2.5
Protein MS Spectra
0
25 m
Time (min)
Protein secondary structure is shown at the top.
hydrogen
Protein
Max = 59 Da
30
Pepsin digestion, 0°C, pH 2.5
40
m/z
35
Workflow4
60
m/z
40
Peak Width (Da)
4h 1476 1480 1484
60
0.1
Continuous labeling hydrogen exchange experiments were performed at room temperature. Labeled samples were injected into a Shimadzu HPLC (LC-20AD) coupled to a Waters Micromass LCT PremierXE Mass Spectrometer for global exchange. Online pepsin digestion was performed (Itk, Btk) and the peptides separated using a custom nanoACQUITY coupled to a Waters Qtof Premier Mass Spectrometer equipped with ESI source for peptide level analysis. Data were analyzed using MassLynxV4.1, PLGS 2.5 and DynamX 2.0 (Waters).
Backbone amides become deuterated
4h
1244 1248 1252 1256
Relative Deuterium Level (Da)
Recombinant Tec family SH3 domains (Tec, Txk, Itk, Btk, Bmx) were overexpressed in E. coli. Protein domains were purified by either nickel affinity and size exclusion chromatography (Tec, Txk, Btk, Bmx) or as a glutathione-S-transferase (GST)-tagged fusion protein (Itk).
Quench exchange reaction at various times, 0°C, pH 2.5
25 m
Relative Deuterium (Da)
4h
METHODS
D2O buffer
25 m
Relative Deuterium (Da)
EX1 Exchange Mechanism4
0.1
HXMS
10 m
5m
Because deuterium incorporation makes the protein heavier, it can be measured with a mass spectrometer. Uptake of deuterium is a function of backbone hydrogen bonding, solvent accessibility, and time—therefore we can uncover protein dynamics using HXMS. Usually, a protein gains mass gradually over time (EX2), but there are unique examples of cooperative unfolding (right), that results in a “jump” in mass. We investigate these EX1 events in the Tec family SH3 domains and related proteins.
Interpretation in light of structure, if known
5m
Relative Deuterium (Da)
Deuterium oxide—colloquially “heavy water”—contains two deuterium atoms, each heavier than the hydrogen in H2O by 1 neutron. Proteins are made up of amino acid chains held together by peptide (amide) bonds. By diluting protein in deuterium oxide (D2O), the backbone amide hydrogens can undergo exchange with deuterium:
Equilibrate 25°C, formulated pH
10 m
1m
10 m
same pH and temperature
3m
9 88 7 66 5 44 3 22 1 00 0.1 0.1
β1
β2-RT Loop
Itk SH3 209-225
Itk SH3 209-225 +3
3m
β3-Distal Loop-β4
EX1 kinetics in:
UND
Mapping of EX1 Regions onto Protein Structure
Deuterium Uptake Plots
Relative Deuterium (Da)
The Src Homology 3 (SH3) domain is an important regulatory domain found in many proteins.1 Using hydrogen exchange mass spectrometry (HXMS), the physical motions of domains such as this can be studied in solution.2 Previously, HXMS studies on the SH3 domains of the Src family tyrosine kinases showed that the native state dynamics of this domain varies on a wide time scale.3 Here we extend our studies to the SH3 domains of the Tec family kinases and characterize their unique dynamics using HXMS. We found that some Tec members, like their Src “cousins” undergo an uncommon slow kinetic reversible unfolding event—a structural phenomenon possibly related to function. We characterize this event (termed EX1 kinetics) and localize unfolding regions within the protein by cutting the protein up into peptides with an aspartic protease, and analyzing the pieces with mass spectrometry. Shifts in these unique dynamics may be further utilized to explore protein-protein interactions, such as HIV accessory protein binding and viral high-jacking of these immune-related regulatory domains.
REFERENCES [1] Zarrinpar, A., R. P. Bhattacharyya, et al. (2003). Sci. STKE 2003(179): re8-. [2] Wales, T. E.; J. R. Engen (2006). Mass Spectrometry Reviews 25(1): 158-170. [3] Wales, T. E.; J. R. Engen (2006. J. Mol. Biol. 357(5): 1592-1604. [4] Marcsisin, S.; J. Engen (2010). Anal. Bioanal. Chem. 397(3): 967-972.
ACKNOWLEDGEMENTS We would like to thank Sreya Tarafdar and the Smithgall lab for providing us with protein, and the Andreotti lab for providing us with the plasmid containing the Itk mouse SH3 domain. We are grateful to the NIH (GM101135), the Northeastern Provost grant, and the Honors Junior-Senior Project Grant for funding.
• The results of this experiment augment previous studies indicating that the SH3 domain is a dynamic domain in solution with varying temporal and spatial degrees of EX1 unfolding. The figure to the right illustrates SH3 kinetics for many SH3 domains, including EX1 half life, degree of EX1 kinetics, and domain phylogeny based on sequence alignment.
Drk (1.2s) Amph2 (1hr) α-Spec (16.5m)
Btk (6.8m) Itk (6.3m)
TEC
Tec Txk
Bmx
