Skip to main content
SpringerLink
Log in

Tryptophan Environment and Functional Characterization of a Kinetically Stable Serine Protease Containing a Polyproline II Fold

  • SHORT COMMUNICATION
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353 nm with excitation at 295 nm indicating tryptophan to be solvent exposed. Upon denaturation with 6 M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360 nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I- and Cs+) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs+ indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs+, indicating charge reorientation after denaturation. No quenching was observed with Cs+ even at pH 1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13 ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.

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
Fig. 6

Abbreviations

Arg:

Arginine

CanPI:

Capsicum annuum pin-II protease inhibitor

CD:

Circular dichroism

GdnHCl:

Guanidine hydrochloride

GuSCN:

Guanidine thiocyanate

PPII fold:

Polyproline II fold

Trp:

Tryptophan

References

  1. Manning M, Colon W (2004) Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward β-sheet structure. Biochemistry 43:11248–11254

    Article  CAS  PubMed  Google Scholar 

  2. Sanchez-Ruiz JM (2010) Protein kinetic stability. Biophys Chem 148:1–15

    Article  CAS  PubMed  Google Scholar 

  3. Rohamare SB, Dixit V, Nareddy PK, Sivaramakrishna D, Swamy MJ, Gaikwad SM (2013) Polyproline fold – in imparting kinetic stability to an alkaline serine endopeptidase. Biochim Biophys Acta (Proteins and Proteomics) 1834:708–716

    Article  CAS  Google Scholar 

  4. Lehrer SS, Leavis PC (1978) Solute quenching of protein fluorescence. Methods Enzymol 49:222–236

    Article  CAS  PubMed  Google Scholar 

  5. Lakowicz EM, Weber G (1973) Quenching of protein fluorescence by oxygen. detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 12:4171–4179

    Article  CAS  PubMed  Google Scholar 

  6. Dixit VS, Pant A (2000) Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124. Biochim Biophys Acta 1523:261–268

    Article  CAS  PubMed  Google Scholar 

  7. Burstein EA, Abornev SM, Reshetnyak YK (2001) Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. decomposition algorithms. Biophys J 81:1699–1709

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Lehrer SS (1971) Solute perturbation of protein fluorescence. The quenching of tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10:3254–3263

    Article  CAS  PubMed  Google Scholar 

  9. Beechem JM, Brand L (1985) Time-resolved fluorescence of proteins. Ann Rev Biochem 54:43–71

    Article  CAS  PubMed  Google Scholar 

  10. Ababou A, Bombarda E (2001) On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins. Prot Sci 10:2102–2113

    Article  CAS  Google Scholar 

  11. James DR, Demmer DR, Steer RP, Verrall RE (1985) Fluorescence lifetime quenching and anisotropy studies of ribonuclease T1. Biochemistry 24:5517–5526

    Article  CAS  PubMed  Google Scholar 

  12. Malinowski DP, Fridovich I (1979) Chemical modification of arginine at the active site of the bovine erythrocyte superoxide dismutase. Biochemistry 18:5909–5917

    Article  CAS  PubMed  Google Scholar 

  13. Mishra M, Joshi RS, Gaikwad S, Gupta VS, Giri AP (2013) Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors. Biochem Biophys Res Commun 430(3):1060–1065

    Article  CAS  PubMed  Google Scholar 

  14. Bostrom M, Tavares FW, Finet S, Skouri-Panet F, Tardieue A, Ninham BW (2005) Why forces between proteins follow different Hofmeister series for pH above and below pI. Biophys Chem 117:217–224

    Article  CAS  PubMed  Google Scholar 

  15. Finet S, Skouri-Panet F, Casselyn M, Bonnete’ F, Tardieu A (2004) The Hofmeister effect as seen by SAXS in protein solutions. Curr Opin Colloid Interface Sci 9:112–116

    Article  CAS  Google Scholar 

  16. Drake AF, Siligardi G, Gibbons WA (1988) Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(L-lysine) and the implications for protein folding and denaturation studies. Biophys Chem 31:143–146

    Article  CAS  PubMed  Google Scholar 

  17. Rucker LA, Creamer TP (2002) Polyproline II helical structure in protein unfolded states: Lysine peptides revisited. Prot Sci 11:980–985

    CAS  Google Scholar 

Download references

Acknowledgments

The authors are thankful to Dr. M. Fernandes, Organic Chemistry Division, NCL, for allowing the use of CD spectropolarimeter. S. R. is supported by Senior Research Fellowship from the CSIR, New Delhi, India.

Author information

Authors and Affiliations

  1. Division of Biochemical Sciences, National Chemical Laboratory, Dr. HomoBhabha Road, Pune, 411008, India

    Sonali B. Rohamare & Sushama M. Gaikwad

Authors
  1. Sonali B. Rohamare
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sushama M. Gaikwad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sushama M. Gaikwad.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supple. Fig. 1

Determination of pKa from pH-activity profile. (DOC 285 kb)

Supple. Fig. 2

Inhibition profile with phenyl glyoxal. (DOC 281 kb)

Supple. Fig. 3

Kinetics of inhibition with CanPI 7 showing uncompetitive inhibition. (DOC 294 kb)

Supplementary Table 1

(DOC 31 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rohamare, S.B., Gaikwad, S.M. Tryptophan Environment and Functional Characterization of a Kinetically Stable Serine Protease Containing a Polyproline II Fold. J Fluoresc 24, 1363–1370 (2014). https://doi.org/10.1007/s10895-014-1445-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-014-1445-5

Keywords

Search

Navigation