BSDB - Experimental results

The table summarizes experimental findings on stretching of proteins at con- stant speed. The first column indicates the name of a protein. The meaning of the abbreviations is as follows: BCA - bovine carbonic anhydrase, ddFLN - Immunoglobulin-like domain 4, 5 (ddFLN4, ddFLN5) from the Dictyostelium discoideum F-actin cross-linker filamin (ddFLN); FN - fibronectin; GFP - green fluorescent protein; TN - tenascin; CAM - Cell adhesion molecules; Mel-CAM - Ig domains of Mel-CAM (melanoma CAM) a protein that is overexpressed in metastatic melanome - under conditions where the disulfide bonds were either left intact or disrupted through reduction (1,4-dithiothreitol, DTT); VACM1 - vascular cell adhesion molecule-1 ConFn - the beginning of the A-band, I48-I54; A60-A65 - the C-zone in the center of the thick filament (AFn); SKIg - the skeletal-specific Ig insert, Sk47-Sk53; PEVK - α-elastin; ConIg - the cinstitutive I-band part of titin, I27-I34. The numbers in round brackets after the name of the protein indicate the amino acids by which one pulls. If the numbers are not provided pulling is by the termini. The second column provides the PDB structure code, if available. The third column gives the typical number of amino acids in the protein. The fourth column is the ex- perimental result for the maximal force. The fifth column specifies the pulling speed. The sixth column indicates a symbol of the CATH structure classifica- tion scheme. The last column lists the references. The values of N refer to the typical sizes of single domains.

Additional comments: The phytochrome protein was unfolded with and with- out the covalently bound tetrapyrrole chromophore: the asterisks indicate the situation with the chromophore. In the case of spectrin R16, the mean force for the short and long elongation events are equal to 60 and 80 pN, respec- tively, at a pulling speed of 3 nm/s. Notice that BCA which posses shallow knot was stretched in two directions: BCA(Cys-253) was stretched from its N-terminus and Cys-253 (replacement of Gln-253 with Cys) to avoid knot tightening under a tensile stress, where BCA_d correspond to engineered dimer of bovine carbonic anhydrase (unit I without knot structure, unit II with knot topology) stretched by N and C-terminal. However, in both constructs the authors of experiments ([64,65]) found that the mechanical stability of this knotted protein was too high to unfold the native state in AFM experiments.

Protein	PDB	N	F_max [pN]	v_p [nm/s]	CATH	References
Titin
I1_I27*4	1g1c	97	127	600	2.60.40.10	[30]
I4		90	171+/26		2.60	[27]
I5		~90	155+/-33		2.60	[27]
I4-I11		~90	150-200+/30		2.60	[27]
I27*8	1tit	89	204+/-30	0.2-1.5 typically 0.6	2.60.40.10	[2,1,3,4,16] [17,18,26,27] [22,29,37,56]
I28		93	257+/-27		2.60.40.10	[26,27,4]
I27-I28		~91	211-306	1	2.60.40.10	[26]
I27-I30		~90	230		2.60.40.10	[2]
I27-I34		~90	150-330+/-20	1	2.60.40.10	[2,26,27]
I27-I34		~90	231+/-6	0.5	2.60.40.10	[48,55]
I32		90	298+/-24		2.60.40.10	[27]
I34		90	281		2.60.40.10	[27]
Sk47-Sk53		~96	210	0.5	2.60	[48]
A60-A65		~99	180	0.5	2.60	[48]
Fibronectins typeIII
pFN		~90	80-200	0.6	2.60	[41,47,50]
¹RNIII	1oww	93	230	0.6	2.60	[41]
¹FNIII_2FNIII		97	220+/-44	0.6	2.60	[41]
²FNIII_1FNIII		91	220+/-44	0.6	2.60	[41]
¹FNIII_I27		97	≤20	0.6	2.60	[41]
¹⁰FNIII	1fnf	~92	74+/-20	0.6	2.60	[29,41]
¹¹FNIII		94	74+/-20	0.6	2.60.40.30	[45]
¹²FNIII_13FNIII	1fnh	92	124+/-18	0.6	2.60.40.30	[41]
¹³FNIII_I27	1fnh	89	89+/-18	0.6	2.60.40.30	[41]
^2-14FNIII		~90	145	0.6	2.60	[41]
Other FN typeIII
tenascin-C		~91	137+/-12	0.3-0.5	2.60.40.30	[40–42]
tenascin-X			150+/-20	0.05-2.0	2.60.40.30	[62]
TNFN*15		~91	113	0.2-0.6	2.60.40.30	[48
TNFNAll	1ten	~90	138+/-12	0.3-0.5	2.60.40.30	[40,41]
TNFNA-D		~91	138	0.3-0.5	2.60.40.30	[40]
I54-I59	1nct	~98	210	0.5-1	2.60.40.10	[55,57]
I48-I54,ConFN		~100	200	0.5	2.60	[48]
Spectrins -monomers
native	1u4q	~106	30+/-5	0.3	1.20.58.60	[32,49]
α-spectrin R16	1aj3	~106	60/80+20	3.0	1.20.58.60	[31]
α-spectrin_{13−18,18−21}	1u4q	~106	55+15	0.3	1.20.58.60	[32,34,49]
β-spectrin₁₋₄	1s35	~106	27-13	0.3		[32–34]
α-actin₁₋₄	1hci	~106	38	0.3	1.20.58.60	[34,49]
-dimers
α,β-spectrin^*			54+/-30			[34]
α-actin₁₋₄			50+/-20		1.20.58.60	[34]
Other
PEVK_I27		186	<20	0.4	4	[27,28,36,54]
N2_I27		572	<20		4	[27,57]
ribonuclease H	1rnh	155	19		3.30.420.10	[18]
E2lip3(N-C)_I27	1qjo	80	15+/-10	0.7	2.40.50.100	[13]
E2lip3(N-41)_I27	1qjo	40	177+/-3	0.7	2.40.50.100	[13]
ankyrin*1	1n11	33	37		3	[8,9]
ankyrin*24	1n11	792	450		3	[8]
Mel-CAM		~100	30		2.60.40.10	[59]
Mel-CAM ^+DTT		~100	41		2.60.40.10	[59]
VACM1 ^+DTT	1vcs	~98	40		2.60.40.10	[60]
ddFLN5	1wlh	100	40-100		2.60	[51]
_I27−I30ddFLN4_I31−I34	1ksr	100	45+/-20	0.2-0.4	2.60	[51–53]
C2A	1dqv	127	60		2.60.40.180	[7]
T4 Lysozyme	1b6i	164	64		1.10.530.40	[58]
barnase_I27	1bnr	108	70	0.1-0.5	3.10.450.30	[12]
calmodulin	1cfc	148	20	0.055	1.10.238.10	[7]
ubiquitin(48-C)*(2-7)	1ubq	28	85+/-20	0.3	3.10.20.90	[5,6]
ubiquitin(N-C))	1ubq	76	203	0.2-0.4	3.10.20.90	[5,6]
ubiquitin	1ubq	76	230+/-34	1	3.10.20.90	[6]
GFP_ddFLN	1b9c	238	104+/-40		2.40.155.10	[19]
GFP_Ig	1b9c	238	104+/-40		2.40.155.10	[19]
GFP(3-212)	1emb	219	130+/-30		2.40.155.10	[20]
GFP(132-212)	1emb	80	120+/-30		2.40.155.10	[20]
GFP(3-132)	1emb	129	350+/-40	3.5	2.40.155.10	[20]
GFP(3-132)	1emb	129	346+/-46	2.0	2.40.155.10	[20]
GFP(3-132)	1emb	129	407+/-45	12.0	2.40.155.10	[20]
GFP(182-212)	1emb	30	356+/-61	3.6	2.40.155.10	[21]
GFP(117-182)	1emb	65	548+/-57	3.6	2.40.155.10	[21]
protein L	1hz6	67	152+/-5	0.7	3.10.20.10	[11]
protein G	1pga	56	190+/-20	0.4	3.10.20.10	[43]
scaffoldin c7A	1aoh	147	480+/-14	0.4		[61]
scaffoldin c1C	1g1k	143	425+/-9	0.4		[61]
scaffoldin c2A	1anu	138	214+/-8	0.4		[61]
spider silk pS(S4+1)		~608	176+/-73	0.2-1.5	2	[46]
filamin A* 24		96	50-220	0.37	2.60	[23]
β2-Microglobulin	1a1m	96	146+/-27	2180	2	[66]
β2-Microglobulin-Δ6N	1a1m	90	80+/-17	2180	2	[66]
Knotted proteins
phytochrome	2o9c	296	47	1		[63]
phytochrome*	2o9c	296	73	1		[63]
BCA II(Cys-253)	1v9e	259	1600*		3.10.200.10	[64]
BCA_d II	1v9e	259	1700		3.10.200.10	[65]
Others
bacteriorhodopsin	1at9	231	350		1.20.1070.10	[44,24]
biotin-streptavidin			350			[38]
proteomer out of hexagonaly packed intermediate layer			312+/-43			[38]
A-macroglobulin			750			[39]
β-fibrils						[25]
DNA			13			[14,15]
P5abc three helix junction			19			[35]

[1] L. Tskhovrebova, K. Trinick, J. A. Sleep, and M. Simmons, Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387 308-312 (1997).

[2] M. Rief, M. Gautel, F. Oesterhelt, J. M. Fernandez, and H. E. Gaub, Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109- 1112 (1997).

[3] M. Carrion-Vasquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, J. M. Fernandez, Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl. Acad. Sci. U.S.A. 96 3694-3699 (1999).

[4] S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, Mechanical Unfolding of a Titin Ig Domain: Structure of Unfolding Intermediate Revealed by Combining AFM, Molecular Dynamics Simulations, NMR and Protein Engineering. J. Mol. Biol. 322 841-849 (2002).

[5] M. Carrion-Vazquez, H. Li, H. Lu, P. E. Marszalek, A. F. Oberhauser, and J. M. Fernandez, The mechanical stability of ubiquitin is linkage dependent. Nat. Struct. Biol. 10 738-743 (2003).

[6] C-L. Chyan, F-C. Lin, H. Peng, J-M. Yuan, C-H. Chang, S-H. Lin, and G. Yang, Reversible mechanical unfolding of single ubiquitin molecules. Biophys. J. 87 3995- 4006 (2004).

[7] M. Carrion-Vazquez, A. F. Oberhauser, T. E. Fisher, P. E. Marszalek, H. Li, and J. M. Fernandez, Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog. Biophys. Mol. Biol. 74 63-91 (2000).

[8] G. Lee, K. Abdi, Y. Jiang, P. Michaely, V. Bennett, P. E. Marszalek, Nanospring behavior of ankyrin repeats. Nature 246-249 (2006).

[9] L.W. Li, S.Wetzel, A. Pluckthun, and J. M. Fernandez, Stepwise unfolding of ankyrin repeats in a single protein revealed by atomic force microscopy. Biophys. J.: 90 L30-32 (2006).

[10] M. T. Alam, T. Yamada, U. Carlsson, A. Ikai, The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase. FEBS Letters 519, 35-40 (2002)

[11] D. J. Brockwell, S. Godfrey, S Beddard, E. Paci, Dan K. West, P. D. Olmsted, D. Alastair Smith, S. E. Radford, Mechanically unfolding small topologically simple protein L. Biophys. J. 89, 506-519 (2005).

[12] R. B. Best, B. Li, A. Steward, V. Daggett, J. Clarke, Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys. J. 81, 2344-2356 (2001).

[13] D. J. Brockwell, E. Paci, R. C. Zinober, G. Beddard, P. D. Olmsted, D. A. Smith, R. N. Perham, S. E. Radford, Pulling geometry defines mechanical resistance of b-sheet protein. Nat. Struct. Biol. 10, 731-737 (2003).

[14] U. Bockelmann, B. Essevaz-Roulet, F. Heslot, Molecular stick-slip motion revealed by opening DNA with piconewton forces. Phys. Rev. Lett. 79, 4489-4492 (1997)

[15] C. G. Baumann, V. A. Bloomfield, S. B. Smith, C. Bustamante, M. D. Wang, S. M. Block, Stretching of single collapsed DNA molecules. Biophys. J. 78, 1965-1978 (2000).

[16] D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, S. E. Radford, The effect of core destabilization on the mechanical resistance of I27. Biophys. J. 83, 458-472 (2002).

[17] M. Carrion-Vazquez, P. E. Marszalek, A. F. Oberhauser, J. M. Fernandez, Atomic force microscopy captures length pheno-types in single proteins. Proc. Natl. Acad. Sci. (USA) 96, 11288-11292 (1999).

[18] C. Cecconi, E. A. Shank, C. Bustamante, S.Marqusee, Direct observation of the threestate folding of a single protein molecule. Science 309, 2057-2060 (2005).

[19] H. Dietz and M. Rief, Exploring the energy landscape of the GFP by single-molecule mechanical experiments. PNAS 101 16192-16197 (2004)

[20] H. Dietz and M. Rief, Protein structure by mechanical triangulation. PNAS 103 1244- 1247 (2006)

[21] Dietz H, Berkemeier F, Bertz M, Rief M. Anisotropic deformation response of single protein molecules. PNAS 103 12724-12728 (2006)

[22] H. P. Erickson, Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin. Proc. Natl. Acad. Sci. (USA) 91, 10114-10118 (1994).

[23] S. Furuike, I. T. Yamazaki M., Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy. FEBS lett. 498, 72-75 (2001).

[24] H. Janovjak, M. Kessler, D. Oesterhelt, H. Gaub, D. J. Muller, Unfolding pathways of native bacteriorhodopsin depend on temperature. EMBO J. 22, 5220-5229 (2003).

[25] M.S.Z. Kellermayer, L. Grama, A. Karsai, A. Nagy, A.Kahn, Z.L. Datki, B. Penke, Reversible mechanical unzipping of Amyloid b-fibrils. J. Biol. Chem. 280, 8464-8470 (2005).

[26] H. Li, A. F. Oberhauser, S. B. Fowler, J. Clarke, J. M. Fernandez, Atomic force microscopy reveals the mechanical design of a modular protein. Proc. Natl. Acad. Sci. (USA) 97, 6527-31 (2000).

[27] H. Li, W. A. Linke, A. F. Oberhauser, M. Carrion-Vazquez, J. G. Kerkviliet, H. Lu, P. E. Marszalek, J. M. Fernandez, Reverse engineering of the giant muscle protein titin. Nature 418, 998-1002 (2002).

[28] H. Li, A. F. Oberhauser, S. D. Redick, M. Carrion-Vazquez, H. P. Erickson, J. M. Fernandez, Multiple conformations of PEVK proteins detected by single-molecule techniques. Proc. Natl. Acad. Sci. (USA) 98, 10682-10686 (2001).

[29] L. Li, H. Han-Li Huang, C. L. Badilla, J. M. Fernandez, Mechanical Unfolding Intermediates Observed by Single-molecule force spectroscopy in fibronectin Type III Module. J.Mol.Biol. 345, 817-826 (2005).

[30] H. B. Li, and J. M. Fernandez, Mechanical design of the first proximal Ig domain of hman cardiac titin revealed by single molecule force spectroscopy. J.Mol.Biol. 334, 75-86 (2003).

[31] P. F. Lenne, A. J. Raae, S. M. Altmann, M. Saraste and J. K. H. Horber, States and transition during unfolding of a single spectrin repeat. FEBS Lett. 476, 124-128, (2000).

[32] R. Law, P. Carl, S.Harper, P. Dalhaimer, D. W. Speicher and D. E. Discher, Cooperativity in force unfolding of tandem spectrin repeats. Biophys. J. 84, 533-544 (2003).

[33] R. Law, G. Liao, S. Harper, G. Yang, D. Speicher and D. E. Discher, Pathway shifts and thermal softening in temerature-coupled forced unfolding of spectrin domains. Biophys. J. 85, 3286-3293 (2003).

[34] R. Law, P. Carl, S. Harper, D. W. Speicher and D. E. Discher, Infulence of lateral association on forced unfolding of antiparallel spectrin heterodimers. J. Biol. Chem. 279, 16410-16416 (2004).

[35] J. Liphardt, B. Onoa, SB Smith, I. Tinoco and C. Bustamante, Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733-737 (2001)

[36] K. Ma, Lou-sing Kan, Kuan Wang, Polyproline II helix is a key structural motif of the elastic PEVK segment of titin. Biochemistry 40, 3427-3438 (2001).

[37] P. E. Marszalek, H. Lu, H. B. Li, M. Carrion-Vazquez, A. F. Oberhauser, K. Schulten, J. M. Fernandez, Mechanical unfolding intermediates in titin modules. Nature 402, 100-103 (1999).

[38] D. J. Muller, W. Baumeister, A. Engel, Controlled un-zipping of a bacterial surface layer with atomic force microscopy. Proc. Natl. Acad. Sci. (USA) 96, 13170-13174 (1999).

[39] K. Mitsui, M. Hara, A. Ikai, Mechanical unfolding of alpha 2-macroglobulin molecules with atomic force microscope. FEBS Lett. 385, 29-33 (1996).

[40] A. F. Oberhauser, P. E. Marszalek, H. P. Erickson, J. M. Fernandez, The molecular elasticity of the extracellular matrix protein tenascin. Nature 14, 181-185 (1998).

[41] A. F. Oberhauser, C. Badilla-Fernandez, M. Carrion-Vazquez, J. M. Fernandez, The mechanical hierarchies of fibronectin observed with single-molecule AFM. J. Mol. Biol. 319, 433-447 (2002).

[42] Y. Cao and H.B. Li, Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin, J. Mol. Biol. 361, 372-381 (2006).

[43] Y. Cao and H.B. Li Polyprotein of GB1 is an ideal artificial elastomeric protein. Nature Mat 6 109-114 (2007).

[44] F. Oesterhelt, D. Oesterhelt, M. Pfeiffer, A. Engel, H. E. Gaub, D. J.Muller, Unfolding pathways of individual bacteriorhodopsins. Science 288, 143-146 (2000).

[45] Y. Oberdorfer, H. Fuchs, A. Janshoff, Conformational analysis of native fibronectin by means of force spectroscopy. Langmuir 16, 9955-9958 (2000).

[46] E. Oroudjev, J. Soares, S. Arcidiacono, J. B. Thompson, S. A. Fossey, and H. G. Hansma, Segmented nanofibers of sider dragline silk: atomic Force microscopy and single-molecule force spectroscopy. Proc. Natl. Acad. Sci. (USA) 30, 6460-6465 (2002).

[47] Y. Oberdoerfer, H. Fusch, A. Janshoff, Conformational analysis of native fibronectin by means of force spectorscopy. Langmuir 16, 9955-9958 (2000).

[48] M. Rief, M. Gautel, M. Schemmel, H. G. Gaub, The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy. Biophys. J. 75, 3008-3014 (1998).

[49] M. Rief, J. Pascual, M. Saraste, H. G. Gaub, Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles. J. Mol. Biol. 286, 553-561 (1999).

[50] M. Rief, M. Gautel, H. E. Gaub, Unfolding forces of titin and fibronectin domains directly measured by AFM. Adv. Exp. Med. Biol. 481, 129-136 (2000).

[51] I. Schwaiger, A. Kardinal, M. Schleicher, A. A. Noegel and M. Rief, A mechanical unfolding intermediate in an actin-crosslinking protein. Nat. Struct. Mol. Biol. 11 81 - 85 (2004).

[52] M. Schlierf and M. Rief , Temperature Softening of a Protein in Single-molecule Experiments. J. Mol. Biol. 354 497-503 (2005).

[53] M. Schlierf and M. Rief, Single-Molecule Unfolding Force Distribution Reveal a Funnel-Shape Energy Landscape. Biophys. J. 90 L33-L35 (2006).

[54] A. Sarkar, S. Caamano, J. M. Fernandez, The elasticity of individual titin PEVK exons measured by single molecule atomic force microscopy. J. Biol. Chem. 280, 6261-6264 (2005).

[55] K. Watanabe, C. Muhle-Goll, M. S. Z. Kellermayer, S. Labeit, H. L. Granzier, Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments. Struct. Biol. J. 137, 248-258 (2002).

[56] P.M.Williams, S. B. Fowler, R. B. Best, J. L. Toca-Herrera, K. A. Scott, A. Steward, J. Clarke, Hidden complexity in the mechanical properties of titin. Nature 422, 446-449 (2003).

[57] K. Watanabe, P. Nair, D. Labeit, M. S. Z. Kellermayer, M. Greaser, S. Labeit, H. L. Granzier, Molecular mechanics of cardiac titins PEVK and N2B spring elements. J. Biol. Chem. 277, 11549-11558 (2002).

[58] G. Yang, C. Cecconi, W. A. Baase, I. R. Vetter, W. A. Breyer, J. A. Haack, B. W. Matthews, F. W. Dahlquist, C. Bustamante, Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme. Proc. Nat. Acad. Sci. (USA) 97, 139-144 (2000).

[59] P. Carl, C. H. Kwok, G. Manderson, D. W. Speicher, D. Discher, Force unfolding modulated by disulphide bonds in the Ig domains of a cell adhesion molecule. Proc. Natl. Acad. Sci. (USA) 98, 1565-1570 (2001).

[60] N. Bhasin, P. Carl, S. Harper, G. Feng, H. Lu, D. W. Speicher and D. E. Discher. Chemistry on a single protein, vascular cell adhesion molecule-1, during forced unfolding. J. Biol. Chem. 279, 45865-45874 (2004).

[61] A. Valbuena, J Oroz, R Hervis, A. M. Vera, D. Rodriguez, M. Menndez, J. I. Sulkowska, M. Cieplak, M. Carrion-Vazquez On the remarkable mechanostability of scaffoldins and the mechanical clamp motif. Proc. Natl. Acad. Sci. (USA) 106 13791- 13796. (2009)

[62] A. Jollymore, C. Lethias, Q. Peng, Y. Cao and H. Li, J. Mol. Biol. 385, 1277-1286, (2009).

[63] T. Bornschloegl, D. M. Anstrom, E. Mey, J. Dzubiella, M. Rief, and K. T. Forest, Tightening the knot in phytochrome by single molecule atomic force microscopy Biophys. J. 96, 1508-1514 (2009).

[64] M. F. Alam and A. Ikai, Protein stretching. V. Two forms of carbonic dehydratase detected by force microscopy. Appl. Phys. A 72, S121-S124 (2000).

[65] T. Wang, H. Arakawa and A. Ikai, Force Measurement and Inhibitor Binding Assay of Monomer and Engineered Dimer of Bovine Carbonic Anhydrase B. Biochemical and Biophysical Research Communications 285, 9-14 (2001).

[66] B. Sorce, S. Sabella, M. Sandal, B. Samori, A. Santino, R. Cingolani, R. Rinaldi and P. P. Pompa Single-Molecule Mechanical Unfolding of Amyloidogenic Beta2-Microglobulin: The Force-Spectroscopy Approach ChemPhysChem 10, 1471-1477 (2009)