Abstract
Methods have been developed to measure the fluorescence lifetime versus temperature of trapped biomolecular ions derivatized with a fluorescent dye. Previous measurements for different sequences of polyproline peptides demonstrated that quenching rates are related to conformations and their spatial fluctuations. This paper presents the results of extending these methods to study the conformational dynamics of larger biomolecules. Vancomycin-peptide noncovalent complexes in the 1+ charge state were studied as a function of temperature for different W-KAA peptide chiralities (L-LDD, D-LDD, L-DLL). Fluorescence-quenching rates, kq, were found to be stereoselective for these different chiralities with relative magnitudes kq(L-LDD)>kq(D-LDD)>kq(L-DLL). The variation in fluorescent quenching resulting from switching the chirality of the single Trp residue was readily detectable. Molecular dynamics analysis of complexes formed by W-KAA (L-LDD) and W-KAA(L-DLL) indicates that increased flexibility in the (L-DLL) complex is correlated with reduced quenching rates. Fluorescence measurements were also performed for the Trp-cage protein comparing quenching rates in the 1+, 2+, and 3+ charge states for which k +q ≫k 2+q ≈k 3+q . Measurements of a sequence including a single-point mutation infer the presence of a salt-bridge structure in the 1+ charge state and its absence in both the 2+ and 3+ states. Molecular dynamics structures of Trp-cage indicate that a salt bridge in the 1+ charge state produces more compact conformations leading to larger quenching rates based on the quenching mechanism. In both these experimental studies the fluorescence-quenching rates were consistent with changes in structure induced by either intermolecular or intramolecular interactions.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Shi, X.; Duft, D.; Parks, J. H. Fluorescence Quenching Induced by Conformational Fluctuations in Unsolvated Polypeptides. J. Phys. Chem. B 2008, 112, 12801–12815.
Molecular Probes. http://probes.invitrogen.com (accessed 10/2009).
Williams, D. H.; Williamson, M. P.; Butcher, D. W.; Hammond, S. J. Detailed Binding Sites of the Antibiotics Vancomycin and Ristocetin A: Determination of Intermolecular Distances in Antibiotic/Substrate Complexes by Use of the Time-Dependent NOE. J. Am. Chem. Soc. 1983, 105, 1332–1339.
Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H. Designing a 20-Residue Protein. Nat. Struct. Biol. 2002, 9, 425–430.
Iavarone, A. T.; Duft, D.; Parks, J. H. Shedding Light on Biomolecule Conformational Dynamics Using Fluorescence Measurements of Trapped Ions. J. Phys. Chem. A 2006, 110, 12714–12727.
(a) http://www.rcsb.org, and PDB code is 1aa5;
Loll, P. J.; Bevivino, A. E.; Korty, B. D.; Axelen, P. H. Simultaneous Recognition of a Carboxylate-Containing Ligand and an Intramolecular Surrogate Ligand in the Crystal Structure of an Asymmetric Vancomycin Dimer. J. Am. Chem. Soc. 1997, 119, 1516–1522.
Maple, J.; Dinur, U.; Hagler, A. T. Derivation of Force Fields for Molecular Mechanics and Dynamics from Ab Initio Energy Surfaces. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5350–5354.
Tsallis, C.; Stariolo, D. A. Generalized Simulated Annealing. Physica A 1996, 233, 395–406.
Iavarone, A. T.; Patriksson, A.; Van der Spoel, D.; Parks, J. H. Fluorescence Probe of Trp-Cage Protein Conformation in Solution and in Gas Phase. J. Am. Chem. Soc. 2007, 129, 6726–6735.
Lindahl, E.; Hess, B.; Van der Spoel, D. GROMACS. 3.0: A Package for Molecular Simulation and Trajectory Analysis. J. Mol. Mod. 2001, 7, 306–317.
Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, Flexible, and Free. J. Comp. Chem. 2005, 26, 1701–1718.
Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225–11236.
Hukushima, K.; Nemoto, K. Exchange Monte Carlo Method and Application to Spin Glass Simulations. J. Phys. Soc. Jpn. 1996, 65, 1604–1608.
Counterman, A. E.; Clemmer, D. E. Anhydrous Polyproline Helices and Globules. J. Phys. Chem. B 2004, 108, 4885–4898.
Perkins, H. R. Specificity of Combination between Mucopeptide Precursors and Vancomycin or Ristocetin. Biochem. J. 1969, 111, 195–205.
Nieto, M.; Perkins, H. R. Modifications of the Acyl-D-Alanyl-D-Alanine Terminus Affecting Complex-Formation with Vancomycin. Biochem. J. 1971, 123, 789–803.
Perkins, H. R. Vancomycin and Related Antibiotics. Pharmacol. Ther. 1982, 16, 181–197.
Rajagopalan, J. S.; Harris, C. M.; Harris, T. M. The Role of Phenolic Groups in Vancomycin-Type Glycopeptide Antibiotics. Bioorg. Chem. 1995, 23, 54–71.
Williams, D. H.; Westwell, M. S. The Fight Against Antibiotic-Resistant Bacteria. Chemtech 1996, 26, 17–23.
Williams, D. H.; Kalman, J. R. Structural and Mode of Action Studies on the Antibiotic Vancomycin: Evidence from 270-MHz Proton Magnetic Resonance. J. Am. Chem. Soc. 1977, 99, 2768–2774.
Loll, P. J.; Miller, R.; Weeks, C. M.; Axelsen, P. H. A Ligand-Mediated Dimerization Mode for Vancomycin. Chem. Biol. 1998, 5, 293–298.
Kaplan, J.; Korty, B. D.; Axelsen, P. H.; Loll, P. J. The Role of Sugar Residues in Molecular Recognition by Vancomycin. J. Med. Chem. 2001, 44, 1837–1840.
Hamdan, M.; Curcuruto, O.; Di Modugno, E. Investigation of Complexes between Some Glycopeptide Antibiotics and Bacterial Cell-Wall Analogues by Electrospray-and Capillary Zone Electrophoresis/Electrospray-Mass Spectrometry. Rapid Commun. Mass Spectrom. 1995, 9, 883–887.
Lim, H. K.; Hsieh, Y. L.; Ganem, B.; Henion, J. Recognition of Cell-Wall Peptide Ligands by Vancomycin Group Antibiotics—Studies Using Ion-Spray Mass-Spectrometry. J. Mass Spectrom. 1995, 30, 708–714.
Vollmerhaus, P. J.; Breukink, E.; Heck, A. J. R. Getting Closer to the Real Bacterial Cell Wall Target: Biomolecular Interactions of Water-Soluble Lipid II with Glycopeptide Antibiotics. Chemistry 2003, 9, 1556–1565.
Heck, A. J. R.; Bonnici, P. J.; Breukink, E.; Morris, D.; Wills, M. Modification and Inhibition of Vancomycin Group Antibiotics by Formaldehyde and Acetaldehyde. Chemistry 2001, 7, 910–916.
Jorgensen, T. J. D.; Delforge, D.; Remacle, J.; Bojesen, G.; Roepstorff, P. Collision-Induced Dissociation of Noncovalent Complexes Between Vancomycin Antibiotics and Peptide Ligand Stereoisomers: Evidence for Molecular Recognition in the Gas Phase. Int. J. Mass Spectrom. 1999, 188, 63–85.
Haselmann, K. F.; Jørgensen, T. J. D.; Budnik, B. A.; Jensen, F.; Zubarev, R. A. Electron Capture Dissociation of Weakly Bound Polypeptide Polycationic Complexes. Rapid Commun. Mass Spectrom. 2002, 16, 2260–2265.
Jørgensen, T. J. D.; Roepstorff, P. Direct Determination of Solution Binding Constants for Noncovalent Complexes between Bacterial Cell Wall Peptide Analogues and Vancomycin Group Antibiotics by Electrospray Ionization Mass Spectrometry. Anal. Chem. 1998, 70, 4427–4432.
Heck, A. J. R.; Jørgensen, T. J. D.; O’Sullivana, M.; Von Raumera, M.; Derrick, P. J. Gas-Phase Noncovalent Interactions between Vancomycin-Group Antibiotics and Bacterial Cell-Wall Precursor Peptides Probed by Hydrogen/Deuterium Exchange. J. Am. Soc. Mass Spectrom. 1998, 9, 1255–1266.
Jørgensen, T. J. D.; Hvelplund, P.; Andersen, J. U.; Roepstorff, P. Tandem Mass Spectrometry of Specific versus Nonspecific Noncovalent Complexes of Vancomycin Antibiotics and Peptide Ligands. Int. J. Mass Spectrom. 2002, 219, 659–670.
Heck, A. J. R.; Jorgensen, T. J. D. Vancomycin in Vacuo. Int. J. Mass Spectrom. 2004, 236, 11–23.
Gerhard, U.; Mackay, J. P.; Maplestone, R. A.; Williams, D. H. The Role of the Sugar and Chlorine Substituents in the Dimerization of Vancomycin Antibiotics. J. Am. Chem. Soc. 1993, 115, 232–237.
These MD simulations were performed by Zhibo Yang, Xiangguo Shi, Erich R. Vorpagel and Julia Laskin at the Pacific Northwest National Laboratory.
Yang, Z.; Vorpagel, E. R.; Laskin, J. Experimental and Theoretical Studies of the Structures and Interactions of Vancomycin Antibiotics with Cell Wall Analogues. J. Am. Chem. Soc. 2008, 130, 13013–13022.
Gellman, S. H.; Woolfson, D. N. Mini-Proteins Trp the Light Fantastic. Nat. Struct. Biol. 2002, 9, 408–410.
Qiu, L.; Pabit, S. A.; Roitberg, A. E.; Hagen, S. J. Smaller and Faster: The 20 Residue Trp-Cage Protein Folds in 4 µs. J. Am. Chem. Soc. 2002, 124, 12952–12953.
Simmerling, C.; Strockbine, B.; Roitberg, A. E. All-Atom Structure Prediction and Folding Simulations of a Stable Protein. J. Am. Chem. Soc. 2002, 124, 11258–11259.
Snow, C. D.; Zagrovic, B.; Pande, V. S. The Trp Cage: Folding Kinetics and Unfolded State Topology Via Molecular Dynamics Simulations. J. Am. Chem. Soc. 2002, 124, 14548–14549.
Pitera, J. W.; Swope, W. Understanding Folding and Design: Replica-Exchange Simulations of “Trp-cage” Miniproteins. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7587–7592.
Chowdhury, S.; Lee, M. C.; Xiong, G. M.; Duan, Y. Ab Initio Folding Simulation of the Trp-Cage Mini-Protein Approaches NMR Resolution. J. Mol. Biol. 2003, 327, 711–717.
Zhou, R. Trp-cage: Folding Free Energy Landscape in Explicit Water. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13280–13285.
Seshasayee, A. S. High-Temperature Unfolding of a Trp-Cage Mini-Protein: A Molecular Dynamics Simulation Study. Theor. Biol. Med. Model. 2005, 2, 7.
Ding, F.; Buldyrev, S. V.; Dokholyan, N. V. Folding Trp-Cage to NMR Resolution Native Structure Using a Coarse-Grained Protein Model. Biophys. J. 2005, 88, 147–155.
Iavarone, A. T.; Parks, J. H. Conformational Change in Unsolvated Trp-Cage Protein Probed by Fluorescence. J. Am. Chem. Soc. 2005, 127, 8606–8607.
Iavarone, A. T.; Meinen, J.; Schulze, S.; Parks, J. H. Fluorescence Probe of Polypeptide Conformational Dynamics in Gas Phase and in Solution. Int. J. Mass Spectrom. 2006, 253, 172–180.
Kjeldsen, F.; Silivra, O. A.; Zubarev, R. A. Zwitterionic States in Gas-Phase Polypeptide Ions Revealed by 157-nm Ultra-Violet Photodissociation. Chem. Eur. J. 2006, 12, 7920–7928.
Patriksson, A.; Adams, C. M.; Kjeldsen, F.; Zubarev, R. A.; Van der Spoel, D. A. Direct Comparison of Protein Structure in the Gas and Solution Phase: The Trp-Cage. J. Phys. Chem. B 2007, 111, 13147–13150.
These MD simulations are being performed by David Van der Spoel at Uppsala University.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online January 25, 2010
Rights and permissions
About this article
Cite this article
Shi, X., Parks, J.H. Fluorescence lifetime probe of biomolecular conformations. J Am Soc Mass Spectrom 21, 707–718 (2010). https://doi.org/10.1016/j.jasms.2010.01.009
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2010.01.009