Conformational exchange in a membrane transport protein is altered in protein crystals.
Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B(12). Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.
Freed
Daniel M
DM
Departments of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
Horanyi
Peter S
PS
Wiener
Michael C
MC
Cafiso
David S
DS
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D058767:Protein Unfolding; D013113:Spin Labels; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D013113:Spin Labels
Nature. 2007 Dec 13;450(7172):964-72
18075575
J Mol Biol. 2007 Jul 27;370(5):807-11
17555764
Science. 1992 May 1;256(5057):655-9
1585178
Trends Biochem Sci. 2008 Jul;33(7):330-8
18539464
Science. 1991 Dec 13;254(5038):1598-603
1749933
Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7):1131-7
12832755
BMC Biochem. 2008 Nov 25;9:31
19032749
Science. 2010 Feb 5;327(5966):653-4
20133562
Mol Pharmacol. 2008 Mar;73(3):880-90
18055762
Chem Phys Lipids. 1994 Sep 6;73(1-2):159-80
8001180
Mol Microbiol. 2003 Aug;49(4):869-82
12890014
FEBS Lett. 2003 Nov 27;555(1):139-43
14630334
Biophys J. 2005 Apr;88(4):2554-63
15681654
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
Proteins. 2001 Jun 1;43(4):509-19
11340666
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11382-7
13679579
Biophys J. 2006 Aug 15;91(4):1441-51
16731567
Biochemistry. 2008 Jan 15;47(2):670-9
18092811
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9518-23
16766663
Biochemistry. 2006 Sep 12;45(36):10847-54
16953570
Science. 2009 Apr 10;324(5924):198-203
19359576
Biochemistry. 2002 Oct 1;41(39):11543-51
12269798
Proteins. 2008 Dec;73(4):802-13
18498104
Biochemistry. 2002 Nov 19;41(46):13473-82
12427007
Nat Struct Biol. 2000 Mar;7(3):205-9
10700278
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W375-83
17452350
Protein Sci. 2010 Feb;19(2):269-78
20014029
Methods Enzymol. 1997;276:307-26
27754618
Proteins. 2000 Aug 15;40(3):389-408
10861930
Curr Opin Struct Biol. 2002 Aug;12(4):431-40
12163064
Curr Opin Struct Biol. 2005 Aug;15(4):394-400
16039843
Proc Natl Acad Sci U S A. 1965 Oct;54(4):1010-7
5219813
Proteins. 2002 Aug 1;48(2):327-40
12112700
Structure. 2006 Feb;14(2):265-74
16472746
Acta Crystallogr D Biol Crystallogr. 2001 Jan;57(Pt 1):122-33
11134934
Biophys J. 1993 Nov;65(5):2097-105
7507718
Curr Opin Struct Biol. 2006 Feb;16(1):102-8
16423525
Biochemistry. 1985 Nov 19;24(24):6756-62
4074726
Am J Physiol. 1999 Jul;277(1):C83-90
10409111
Curr Opin Struct Biol. 2007 Dec;17(6):633-40
18024008
Mol Cell. 2008 Sep 26;31(6):896-908
18922471
Biochemistry. 2003 Nov 18;42(45):13106-12
14609320
Methods Enzymol. 1995;259:43-94
8538466
Nature. 2007 Dec 6;450(7171):913-6
18026087
Protein Sci. 2008 Feb;17(2):228-39
18096642
Biochemistry. 2003 Feb 18;42(6):1391-400
12578351
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
12393927
Biophys J. 2006 Apr 15;90(8):2922-9
16443663
Nat Struct Biol. 2003 May;10(5):394-401
12652322
Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11975-80
17606918
Biophys J. 1985 Mar;47(3):411-4
3978211
Biophys J. 1990 May;57(5):1049-64
2340341