Skip to main content
SpringerLink
Log in

Relaxation dispersion NMR spectroscopy for the study of protein allostery

  • Review
  • Published:
Biophysical Reviews Aims and scope Submit manuscript

Abstract

Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Baldwin AJ (2014) An exact solution for R2, eff in CPMG experiments in the case of two site chemical exchange. J Magn Reson 244:114–124

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensembles in biomolecular recognition. Nat Chem Biol 5:789–796

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bruschweiler S, Schanda P, Kloiber K, Brutscher B, Kontaxis G, Konrat R, Tollinger M (2009) Direct observation of the dynamic process underlying allosteric signal transmission. J Am Chem Soc 131:3063–3068

    Article  CAS  PubMed  Google Scholar 

  • Carver JP, Richards RE (1972) A general two-site solution for the chemial exchange produced dependence of T2 upon the Carr-Purcell pulse separation. J Magn Reson 6:89–105

    CAS  Google Scholar 

  • Chang CP, Shen WF, Rozenfeld S, Lawrence HJ, Largman C, Cleary ML (1995) Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev 9:663–674

    Article  CAS  PubMed  Google Scholar 

  • Cornilescu G, Bax A (2000) Measurement of proton, nitrogen, and carbonyl chemical shielding anisotropies in a protein dissolved in a dilute liquid crystalline phase. J Am Chem Soc 122:10143–10154

    Article  CAS  Google Scholar 

  • Farber PJ, Mittermaier A (2011) Concerted dynamics link allosteric sites in the PBX homeodomain. J Mol Biol 405:819–830

    Article  CAS  PubMed  Google Scholar 

  • Farber P, Darmawan H, Sprules T, Mittermaier A (2010) Analyzing protein folding cooperativity by differential scanning calorimetry and NMR spectroscopy. J Am Chem Soc 132:6214–6222

    Article  CAS  PubMed  Google Scholar 

  • Farber PJ, Slager J, Mittermaier AK (2012) Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data. J Phys Chem B 116:10317–10329

  • Farrow NA, Zhang O, Forman-Kay JD, Kay LE (1994) A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J Biomol NMR 4:727–734

    Article  CAS  PubMed  Google Scholar 

  • Farrow NA, Zhang O, Szabo A, Torchia DA, Kay LE (1995) Spectral density function mapping using 15N relaxation data exclusively. J Biomol NMR 6:153–162

    Article  CAS  PubMed  Google Scholar 

  • Green NC, Rambaldi I, Teakles J, Featherstone MS (1998) A conserved C-terminal domain in PBX increases DNA binding by the PBX homeodomain and is not a primary site of contact for the YPWM motif of HOXA1. J Biol Chem 273:13273–13279

    Article  CAS  PubMed  Google Scholar 

  • Grey MJ, Wang C, Palmer AG 3rd (2003) Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling. J Am Chem Soc 125:14324–14335. This presents CPMG relaxation dispersion equations for systems undergoing linear three-state exchange in the fast timescale regime. Simulations show that when the two exchange pathways differ by about an order of magnitude in timescale, relaxation dispersion profiles are clearly biphasic. As described in this review, biphasic dispersion profiles can manifest as offsets between CPMG- and LOSMQ-derived 15N transverse relaxation rates, R2

    Article  CAS  PubMed  Google Scholar 

  • Hansen DF, Yang D, Feng H, Zhou Z, Wiesner S, Bai Y, Kay LE (2007) An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange. J Am Chem Soc 129:11468–11479. This describes a suite of four NMR spin relaxation experiments that yield 15N transverse relaxation rates that are free from conformational exchange contributions. These can be combined with additional spin relaxation experiments and CPMG data to characterize microsecond timescale and multi-site exchange processes

    Article  CAS  PubMed  Google Scholar 

  • Hansen AF, Vallurupalli P, Kay LE (2008a) An improved N-15 relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904. This describes an 15N CPMG experiment in which 1H decoupling applied during the 15N relaxation delay provides several advantages. Firstly, the experiment is capabale of characterizing slow (≈5-50 s-1) motions with greater accuracy. Secondly, unlike previous CPMG experiments, it measures true 15N transverse relaxation rates that may be compared quantitively to those obtained from different NMR techniques

    Article  CAS  PubMed  Google Scholar 

  • Hansen DF, Vallurupalli P, Kay LE (2008b) An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904

    Article  CAS  PubMed  Google Scholar 

  • Ishima R, Torchia DA (2006) Accuracy of optimized chemical-exchange parameters derived by fitting CPMG R2 dispersion profiles when R2(0a) not = R2(0b). J Biomol NMR 34:209–219

    Article  CAS  PubMed  Google Scholar 

  • Knoepfler PS, Kamps MP (1995) The pentapeptide motif of Hox proteins is required for cooperative DNA-binding with Pbx1, physically contacts Pbx1 and enhances DNA-binding by Pbx1. Mol Cell Biol 15:5811–5819

    PubMed Central  CAS  PubMed  Google Scholar 

  • Korzhnev DM, Salvatella X, Vendruscolo M, Di Nardo AA, Davidson AR, Dobson CM, Kay LE (2004) Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430:586–590

    Article  CAS  PubMed  Google Scholar 

  • Koshland DE Jr, Nemethy G, Filmer D (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365–385

    Article  CAS  PubMed  Google Scholar 

  • Kroenke CD, Loria JP, Lee LK, Rance M, Palmer AG (1998) Longitudinal and transverse H-1-N-15 dipolar N-15 chemical shift anisotropy relaxation interference: Unambiguous determination of rotational diffusion tensors and chemical exchange effects in biological macromolecules. J Am Chem Soc 120:7905–7915

    Article  CAS  Google Scholar 

  • Kumar S, Ma B, Tsai CJ, Sinha N, Nussinov R (2000) Folding and binding cascades: dynamic landscapes and population shifts. Protein Sci 9:10–19

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lefstin JA, Yamamoto KR (1998) Allosteric effects of DNA on transcriptional regulators. Nature 392:885–888

    Article  CAS  PubMed  Google Scholar 

  • Loria JP, Rance M, Palmer AG 3rd (1999) Transverse-relaxation-optimized (TROSY) gradient-enhanced triple-resonance NMR spectroscopy. J Magn Reson 141:180–184

    Article  CAS  PubMed  Google Scholar 

  • Luz Z, Meiboom S (1963) Nuclear magnetic resonance study of protolysis of trimethylammonium ion in aqueous solution-order of reaction with respect to solvent. J Chem Phys 39:366–370

    Article  CAS  Google Scholar 

  • McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430–431

    Article  CAS  Google Scholar 

  • Monod J, Wyman J, Changeux JP (1965) On the nature of allosteric transitions: a plausible model. J Mol Biol 12:88–118

    Article  CAS  PubMed  Google Scholar 

  • Mulder FA, Mittermaier A, Hon B, Dahlquist FW, Kay LE (2001) Studying excited states of proteins by NMR spectroscopy. Nat Struct Biol 8:932–935

    Article  CAS  PubMed  Google Scholar 

  • Neudecker P, Korzhnev DM, Kay LE (2006) Assessment of the effects of increased relaxation dispersion data on the extraction of 3-site exchange parameters characterizing the unfolding of an SH3 domain. J Biomol NMR 34:129–135

    Article  CAS  PubMed  Google Scholar 

  • Nussinov R, Tsai CJ, Xin F, Radivojac P (2012) Allosteric post-translational modification codes. Trends Biochem Sci 37:447–455

    Article  CAS  PubMed  Google Scholar 

  • Nussinov R, Ma B, Tsai CJ, Csermely P (2013) Allosteric conformational barcodes direct signaling in the cell. Structure 21:1509–1521

    Article  CAS  PubMed  Google Scholar 

  • Palmer AG 3rd, Massi F (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev 106:1700–1719

    Article  CAS  PubMed  Google Scholar 

  • Palmer AG, Kroenke CD, Loria JP (2001) NMR methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Meth Enzymol 339:204–238

    Article  CAS  PubMed  Google Scholar 

  • Piper DE, Batchelor AH, Chang CP, Cleary ML, Wolberger C (1999) Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation. Cell 96:587–597

    Article  CAS  PubMed  Google Scholar 

  • Popovych N, Sun S, Ebright RH, Kalodimos CG (2006) Dynamically driven protein allostery. Nat Struct Mol Biol 13:831–838

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Smock RG, Gierasch LM (2009) Sending signals dynamically. Science 324:198–203

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sprules T, Green N, Featherstone M, Gehring K (2003) Lock and key binding of the HOX YPWM peptide to the PBX homeodomain. J Biol Chem 278:1053–1058

    Article  CAS  PubMed  Google Scholar 

  • Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–U1011

  • Tzeng SR, Kalodimos CG (2009) Dynamic activation of an allosteric regulatory protein. Nature 462:368–372

    Article  CAS  PubMed  Google Scholar 

  • Tzeng SR, Kalodimos CG (2013) Allosteric inhibition through suppression of transient conformational states. Nat Chem Biol 9:462–465

    Article  CAS  PubMed  Google Scholar 

  • Vallurupalli P, Bouvignies G, Kay LE (2012) Studying “invisible” excited protein states in slow exchange with a major state conformation. J Am Chem Soc 134:8148–8161

    Article  CAS  PubMed  Google Scholar 

  • Volkman BF, Lipson D, Wemmer DE, Kern D (2001) Two-state allosteric behavior in a single-domain signaling protein. Science 291:2429–2433

    Article  CAS  PubMed  Google Scholar 

  • Wang C, Grey MJ, Palmer AG 3rd (2001) CPMG sequences with enhanced sensitivity to chemical exchange. J Biomol NMR 21:361–366

    Article  CAS  PubMed  Google Scholar 

Download references

Compliance with Ethical Standards

Funding

This research was supported by the National Science and Engineering Research Council (NSERC, Canada, grant number 327028-09). A.M. is a member of Groupe de Recherche Axé sur la Structure des Protéines (GRASP). NMR experiments wererecorded at the Québec/Eastern Canada High Field NMR Facility, supported by McGill University and GRASP.

Conflict of interest

Anthony Mittermaier declares that he has no conflict of interest. Patrick Farber declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Author information

Authors and Affiliations

  1. Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada

    Patrick J. Farber

  2. Department of Chemistry, McGill University, 801 Sherbrooke St. W. room 322, Montreal, Quebec, Canada, H3A 0B8

    Anthony Mittermaier

Authors
  1. Patrick J. Farber
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anthony Mittermaier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anthony Mittermaier.

Additional information

This article is part of a Special Issue on ‘The Role of Protein Dynamics in Allosteric Effects’ edited by Gordon Roberts.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Farber, P.J., Mittermaier, A. Relaxation dispersion NMR spectroscopy for the study of protein allostery. Biophys Rev 7, 191–200 (2015). https://doi.org/10.1007/s12551-015-0166-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12551-015-0166-6

Keywords

Search

Navigation