Skip to main content
SpringerLink
Log in

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.

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

  • Bai Y, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17:75–86

    Article  Google Scholar 

  • Bai Y, Milne JS, Mayne L, Englander SW (1994) Protein stability parameters measured by hydrogen exchange. Proteins 20:4–14

    Article  Google Scholar 

  • Bai Y, Sosnick TR, Mayne L, Englander SW (1995) Protein folding intermediates: native-state hydrogen exchange. Science 269:192–197

    Article  ADS  Google Scholar 

  • Barbar E, Kleinman B, Imhoff D, Li M, Hays TS, Hare M (2001) Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein. Biochemistry 40:1596–1605

    Article  Google Scholar 

  • Becktel WJ, Schellman JA (1987) Protein stability curves. Biopolymers 26:1859–1877

    Article  Google Scholar 

  • Brorsson AC, Kjellson A, Aronsson G, Sethson I, Hambraeus C, Jonsson BH (2004) The “two-state folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange. J Mol Biol 340:333–344

    Article  Google Scholar 

  • Chamberlain AK, Marqusee S (1997) Touring the landscapes: partially folded proteins examined by hydrogen exchange. Structure 5:859–863

    Article  Google Scholar 

  • Chi YH, Kumar TK, Chiu IM, Yu C (2002) Identification of rare partially unfolded states in equilibrium with the native conformation in an all beta-barrel protein. J Biol Chem 277:34941–34948

    Article  Google Scholar 

  • Chu R, Pei W, Takei J, Bai Y (2002) Relationship between the native-state hydrogen exchange and folding pathways of a four-helix bundle protein. Biochemistry 41:7998–8003

    Article  Google Scholar 

  • Dill KA (1990) Dominant forces in protein folding. Biochemistry 29:7133–7155

    Article  Google Scholar 

  • Dyson HJ, Wright PE (1996) Insights into protein folding from NMR. Annu Rev Phys Chem 47:369–395

    Article  Google Scholar 

  • Fuhrmann JC, Kins S, Rostaing P, El FO, Kirsch J, Sheng M, Triller A, Betz H, Kneussel M (2002) Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes. J Neurosci 22:5393–5402

    Google Scholar 

  • Hoang L, Bedard S, Krishna MM, Lin Y, Englander SW (2002) Cytochrome c folding pathway: kinetic native-state hydrogen exchange. Proc Natl Acad Sci USA 99:12173–12178

    Article  ADS  Google Scholar 

  • Honig B (1999) Protein folding: from the levinthal paradox to structure prediction. J Mol Biol 293:283–293

    Article  Google Scholar 

  • Hvidt A, Nielsen SO (1966) Hydrogen exchange in proteins. Adv Protein Chem 21:287–386

    Article  Google Scholar 

  • Jaffrey SR, Snyder SH (1996) PIN: an associated protein inhibitor of neuronal nitric oxide synthase. Science 274:774–777

    Article  ADS  Google Scholar 

  • Kamal JK, Nazeerunnisa M, Behere DV (2002) Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters. J Biol Chem 277:40717–40721

    Article  Google Scholar 

  • Kauzmann W (1959) Some factors in the interpretation of protein denaturation. Adv Protein Chem 14:1–63

    Article  Google Scholar 

  • Krishna Mohan PM (2007) Unfolding energetics and conformational stability of DLC8 monomer. Biochimie 89:1409–1415

    Article  Google Scholar 

  • Krishna Mohan PM, Hosur RV (2007) NMR insights into dynamics regulated target binding of DLC8 dimer. Biochem Biophys Res Commun 355:950–955

    Article  Google Scholar 

  • Krishna Mohan PM, Barve M, Chatterjee A, Ghosh-Roy A, Hosur RV (2008) NMR comparison of the native energy landscapes of DLC8 dimer and monomer. Biophys Chem 134:10–19

    Article  Google Scholar 

  • Krishna MM, Hoang L, Lin Y, Englander SW (2004) Hydrogen exchange methods to study protein folding. Methods 34:51–64

    Article  Google Scholar 

  • Liang J, Jaffrey SR, Guo W, Snyder SH, Clardy J (1999) Structure of the PIN/LC8 dimer with a bound peptide. Nat Struct Biol 6:735–740

    Article  Google Scholar 

  • Linderstrøm-Lang K (1958) Deuterium exchange and protein structure. In: Neuberger (ed) Symposium on protein structure, London

  • Lo KW, Kan HM, Chan LN, Xu WG, Wang KP, Wu Z, Sheng M, Zhang M (2005) The 8-kDa dynein light chain binds to p53-binding protein 1 and mediates DNA damage-induced p53 nuclear accumulation. J Biol Chem 280:8172–8179

    Article  Google Scholar 

  • Lopez MM, Makhatadze GI (2002) Differential scanning calorimetry. Methods Mol Biol 173:113–119

    Google Scholar 

  • Maity H, Maity M, Krishna MM, Mayne L, Englander SW (2005) Protein folding: the stepwise assembly of foldon units. Proc Natl Acad Sci USA 102:4741–4746

    Article  ADS  Google Scholar 

  • Mohan PM, Hosur RV (2008a) NMR characterization of structural and dynamics perturbations due to a single point mutation in Drosophila DLC8 dimer: functional implications. Biochemistry 47:6251–6259

    Article  Google Scholar 

  • Mohan PM, Hosur RV (2008b) pH dependent unfolding characteristics of DLC8 dimer: residue level details from NMR. Biochim Biophys Acta 1784:1795–1803

    Google Scholar 

  • Mohan PM, Barve M, Chatterjee A, Hosur RV (2006) pH driven conformational dynamics and dimer-to-monomer transition in DLC8. Protein Sci 15:335–342

    Article  Google Scholar 

  • Mohan PM, Chakraborty S, Hosur RV (2009a) Residue-wise conformational stability of DLC8 dimer from native-state hydrogen exchange. Proteins 75(1):40–52

    Article  Google Scholar 

  • Mohan PM, Joshi MV, Hosur RV (2009b) Hierarchy in guanidine unfolding of DLC8 dimer: regulatory functional implications. Biochimie 91(3):401–407

    Article  Google Scholar 

  • Mukherjee S, Mohan PM, Kuchroo K, Chary KV (2007) Energetics of the native energy landscape of a two-domain calcium sensor protein: distinct folding features of the two domains. Biochemistry 46:9911–9919

    Article  Google Scholar 

  • Pace CN (1975) The stability of globular proteins. CRC Crit Rev Biochem 3:1–43

    Article  Google Scholar 

  • Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131:266–280

    Article  Google Scholar 

  • Pace CN, Shirley BA, McNutt M, Gajiwala K (1996) Forces contributing to the conformational stability of proteins. FASEB J 10:75–83

    Google Scholar 

  • Privalov PL, Khechinashvili NN (1974) A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol 86:665–684

    Article  Google Scholar 

  • Puthalakath H, Villunger A, O’Reilly LA, Beaumont JG, Coultas L, Cheney RE, Huang DC, Strasser A (2001) Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis. Science 293:1829–1832

    Article  ADS  Google Scholar 

  • Rose GD, Wolfenden R (1993) Hydrogen bonding, hydrophobicity, packing, and protein folding. Annu Rev Biophys Biomol Struct 22:381–415

    Article  Google Scholar 

  • Rumbley J, Hoang L, Mayne L, Englander SW (2001) An amino acid code for protein folding. Proc Natl Acad Sci USA 98:105–112

    Article  ADS  Google Scholar 

  • Shih P, Holland DR, Kirsch JF (1995) Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules. Protein Sci 4:2050–2062

    Article  Google Scholar 

  • Song Y, Benison G, Nyarko A, Hays TS, Barbar E (2007) Potential role for phosphorylation in differential regulation of the assembly of dynein light chains. J Biol Chem 282:17272–17279

    Article  Google Scholar 

  • Stowell MH, Rees DC (1995) Structure and stability of membrane proteins. Adv Protein Chem 46:279–311

    Article  Google Scholar 

  • Vadlamudi RK, Bagheri-Yarmand R, Yang Z, Balasenthil S, Nguyen D, Sahin AA, den HP, Kumar R (2004) Dynein light chain 1, a p21-activated kinase 1-interacting substrate, promotes cancerous phenotypes. Cancer Cell 5:575–585

    Article  Google Scholar 

  • Zipp A, Kauzmann W (1973) Pressure denaturation of metmyoglobin. Biochemistry 12:4217–4228

    Article  Google Scholar 

Download references

Acknowledgments

We thank Government of India for providing financial support to the National Facility for High Field NMR at the Tata Institute of Fundamental Research. The authors acknowledge Dr. Anindya Ghosh-Roy for the DLC8 clone, Dr. M. M. G Krishna for HX Excel spread sheet, and Dr. Sulakshana Mukherjee for critical comments. PMKM is a recipient of TIFR Alumni Association Scholarship (2003–2005) and Sarojini Damodaran International Fellowship for career development, supported by the TIFR endowment fund.

Author information

Authors and Affiliations

  1. Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India

    P. M. Krishna Mohan, Swagata Chakraborty & Ramakrishna V. Hosur

Authors
  1. P. M. Krishna Mohan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Swagata Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ramakrishna V. Hosur
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramakrishna V. Hosur.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(PDF 69 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krishna Mohan, P.M., Chakraborty, S. & Hosur, R.V. NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange. J Biomol NMR 44, 1–11 (2009). https://doi.org/10.1007/s10858-009-9311-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-009-9311-5

Keywords

Search

Navigation