Ultraviolet resonance raman study of the avidin biotin complex
3
JOURNAL OF RAMAN SPECTROSCOPY J. Raman Spectrosc. 31, 373–375 (2000) Ultraviolet resonance Raman study of the avidin biotin complex John Clarkson,* Caroline Sudworth, Sergiu I. Masca, David N. Batchelder and D. Alastair Smith Department of Physics and Astronomy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5, performed with 244 nm excitation, resolves spectral contributions from the tyrosine 33 and tryptophan residues. The UVRR difference spectrum of holo avidin minus apo avidin, reveals details of the changes to avidin’s tyrosine 33. No change in the hydrogen bonding state of tyrosine 33 is detected, but a change to a more hydrophobic environment is indicated upon addition of biotin. Tryptophan bands dominate the difference spectrum, with changes corresponding to the movement of tryptophan tortional angles being observed. The increase in tryptophan UVRR bands upon addition of biotin indicates a change to a more hydrophobic environment, highlighting the importance of hydrophobic interactions in the observed strong avidin/biotin binding. Copyright 2000 John Wiley & Sons, Ltd. INTRODUCTION Biotin forms one of the strongest known protein substrate complexes with avidin, a hen egg white protein. This very high binding affinity has resulted in the complex being exploited in numerous biotechnological applications. 1,2 Hydrogen bonding to the ureido part of biotin is known to play an important role in the interaction. Chemical modi- fication of the single tyrosine residue, Tyr33, results in the complete loss of biotin binding, as does chemical modifi- cation of the tryptophan side groups, Trp 70 or Trp 110. 3,4 The fluorescence of avidin Trp residues is also modified by biotin binding, showing a 34% reduction in quantum efficiency in the complex. 5,6 Ultraviolet absorbance spec- tral changes have been interpreted as evidence that the Trp residues move into a more hydrophobic environment upon biotin binding. 7 Here we present the first reported results from an ultraviolet resonance Raman (UVRR) spectroscopic study of the binding of biotin by avidin. Previous reported visible and near-infrared Raman studies of avidin/biotin provided evidence of secondary structural changes to avidin. An increase in the intensity of bands assigned to Trp residues upon biotin binding was also observed. 8–11 The UVRR study presented here, using 244 nm radiation, provides selective enhancement of the Trp and Tyr Raman signals. 12 This enables unambiguous assignment of the changes to Tyr33 and the Trp residues upon complex formation, revealing further details regarding the nature of the reorientation of the binding pocket of avidin upon addition of biotin. EXPERIMENTAL Avidin and biotin where purchased from Sigma. A solu- tion of 200 μM avidin was prepared in PBS buffer * Correspondence to: J. Clarkson, Department of Physics and Astron- omy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK; e-mail: [email protected] at pH 7.5 containing 5 mM KNO 3 , as an internal Raman intensity standard. Raman spectra were obtained using 2 mW, 244 nm radiation from an intracavity wavenumber-doubled argon ion laser (Coherent Innova 300 FReD). A Renishaw micro-Raman System 1000 spectrometer modified for use at 244 nm was used to acquire the spectra, 13 which were accumulated over 30 minutes, (60 ð 30 seconds). To avoid photodegrada- tion the protein solution was rapidly circulated through a quartz capillary tube, (i.d. D 0.2 mm) by a peri- staltic pump (tubing i.d. D 0.5 mm) from a reser- voir at 4 ° C. Volumes as low as 200 μL could be used with this apparatus. To minimize dilution of the pro- tein solution, solid biotin, approximately 10 times in excess of avidin, was added to one aliquot of avidin solution, gently shaken for 5 minutes and kept on ice for 1 hour. RESULTS AND DISCUSSION Figure 1(a) shows the UVRR spectrum of apo avidin, (no biotin present), at 244 nm. (The spectral features in Fig. 1 are assigned according to previous reports of UVRR studies of proteins). 12,14 The selective enhance- ment of the Trp and Tyr residues is evident and the amide I band at 1662 cm 1 is also reasonably strong. Phe is not resonantly enhanced at this wavelength and does not contribute to the spectra, despite avidin having seven Phe residues. A similar result has been reported for the UVRR spectrum of lysozyme with 244 nm excitation. 15 Upon binding of biotin a number of spectral changes occur in the Tyr and Trp intensities and peak positions, [Fig. 1(b)], that are highlighted in the difference spec- trum, [Fig. 1(b-a)]. Biotin does not exhibit measurable Raman bands at 244 nm, as it is a very weak Raman scatterer, with no electronic absorbance near 244 nm and makes no contribution to the difference spectrum. We shall consider the interpretation of these data in terms of the effects on Tyr33 and the Trp residues separately. Copyright 2000 John Wiley & Sons, Ltd. Received 15 September 1999 Accepted 15 October 1999
-
Upload
john-clarkson -
Category
Science
-
view
225 -
download
3
Transcript of Ultraviolet resonance raman study of the avidin biotin complex
JOURNAL OF RAMAN SPECTROSCOPYJ. Raman Spectrosc. 31, 373–375 (2000)
Ultraviolet resonance Raman study of the avidinbiotin complex
John Clarkson,* Caroline Sudworth, Sergiu I. Masca, David N. Batchelder and D. Alastair SmithDepartment of Physics and Astronomy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK
An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5, performedwith 244 nm excitation, resolves spectral contributions from the tyrosine 33 and tryptophan residues. TheUVRR difference spectrum of holo avidin minus apo avidin, reveals details of the changes to avidin’styrosine 33. No change in the hydrogen bonding state of tyrosine 33 is detected, but a change to a morehydrophobic environment is indicated upon addition of biotin. Tryptophan bands dominate the differencespectrum, with changes corresponding to the movement of tryptophan tortional angles being observed. Theincrease in tryptophan UVRR bands upon addition of biotin indicates a change to a more hydrophobicenvironment, highlighting the importance of hydrophobic interactions in the observed strong avidin/biotinbinding. Copyright 2000 John Wiley & Sons, Ltd.
INTRODUCTION
Biotin forms one of the strongest known protein substratecomplexes with avidin, a hen egg white protein. This veryhigh binding affinity has resulted in the complex beingexploited in numerous biotechnological applications.1,2
Hydrogen bonding to the ureido part of biotin is known toplay an important role in the interaction. Chemical modi-fication of the single tyrosine residue, Tyr33, results in thecomplete loss of biotin binding, as does chemical modifi-cation of the tryptophan side groups, Trp 70 or Trp 110.3,4
The fluorescence of avidin Trp residues is also modifiedby biotin binding, showing a 34% reduction in quantumefficiency in the complex.5,6 Ultraviolet absorbance spec-tral changes have been interpreted as evidence that theTrp residues move into a more hydrophobic environmentupon biotin binding.7
Here we present the first reported results from anultraviolet resonance Raman (UVRR) spectroscopic studyof the binding of biotin by avidin. Previous reportedvisible and near-infrared Raman studies of avidin/biotinprovided evidence of secondary structural changes toavidin. An increase in the intensity of bands assigned toTrp residues upon biotin binding was also observed.8 – 11
The UVRR study presented here, using 244 nm radiation,provides selective enhancement of the Trp and Tyr Ramansignals.12 This enables unambiguous assignment of thechanges to Tyr33 and the Trp residues upon complexformation, revealing further details regarding the natureof the reorientation of the binding pocket of avidin uponaddition of biotin.
EXPERIMENTAL
Avidin and biotin where purchased from Sigma. A solu-tion of 200 µM avidin was prepared in PBS buffer
* Correspondence to: J. Clarkson, Department of Physics and Astron-omy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK;e-mail: [email protected]
at pH 7.5 containing 5 mM KNO3, as an internalRaman intensity standard. Raman spectra were obtainedusing 2 mW, 244 nm radiation from an intracavitywavenumber-doubled argon ion laser (Coherent Innova300 FReD). A Renishaw micro-Raman System 1000spectrometer modified for use at 244 nm was usedto acquire the spectra,13 which were accumulated over30 minutes, (60ð 30 seconds). To avoid photodegrada-tion the protein solution was rapidly circulated througha quartz capillary tube, (i.d.D 0.2 mm) by a peri-staltic pump (tubing i.d.D 0.5 mm) from a reser-voir at 4°C. Volumes as low as 200µL could be usedwith this apparatus. To minimize dilution of the pro-tein solution, solid biotin, approximately 10 times inexcess of avidin, was added to one aliquot of avidinsolution, gently shaken for 5 minutes and kept on icefor 1 hour.
RESULTS AND DISCUSSION
Figure 1(a) shows the UVRR spectrum of apo avidin,(no biotin present), at 244 nm. (The spectral featuresin Fig. 1 are assigned according to previous reports ofUVRR studies of proteins).12,14 The selective enhance-ment of the Trp and Tyr residues is evident and theamide I band at 1662 cm�1 is also reasonably strong. Pheis not resonantly enhanced at this wavelength and doesnot contribute to the spectra, despite avidin having sevenPhe residues. A similar result has been reported for theUVRR spectrum of lysozyme with 244 nm excitation.15
Upon binding of biotin a number of spectral changesoccur in the Tyr and Trp intensities and peak positions,[Fig. 1(b)], that are highlighted in the difference spec-trum, [Fig. 1(b-a)]. Biotin does not exhibit measurableRaman bands at 244 nm, as it is a very weak Ramanscatterer, with no electronic absorbance near 244 nmand makes no contribution to the difference spectrum.We shall consider the interpretation of these data interms of the effects on Tyr33 and the Trp residuesseparately.
Copyright 2000 John Wiley & Sons, Ltd. Received 15 September 1999Accepted 15 October 1999
374 J. CLARKSONET AL.
Figure 1. UVRR spectrum of (a) apo avidin, (b) holo avidin (biotinpresent), and (b a) the difference spectrum. The intensities of(a) and (b) are normalized to the internal standard NO�3 peak at1047 cm�1 and the difference spectrum is scaled by a factor of 2for clarity. The asterisk indicates cosmic ray interference.
Tyrosine 33
The derivative features in the difference spectrum (pairsof positive and negative peaks) at 1604/1617 cm�1 and1159/1174 cm�1 are assigned to upshifts of the Y8a andY9a bands, respectively. Similar features in the UVRRdifference spectra associated with structural changes inbacteriorhodopsin, octopus rhodopsin and haemoglobinhave been reported.16 – 19 An intensity increase in the tryp-tophan W1 peak is likely to contribute to the 1617 cm�1
feature in the difference spectrum. The increase in theintensity of Y7a peak, however, suggests that the differ-ence feature at 1617 cm�1 has a large contribution froma shift and increase in intensity of Y8a. The Y8a peak oftyrosine is more than twice as intense as Y7a.
The UVRR intensities and wavenumbers of Y8a, Y8b,Y7a and Y9a Raman bands are reported to be sensitiveto hydrogen bonding and hydrophobic effects.19,20 Thenoticeable increase in the intensity of Y7a in the differencespectrum, indicates a more hydrophobic environment forY33 upon the addition of biotin. The other tyrosine bandsat 857 and 829 cm�1 in the difference spectrum are due toan increase in the intensity of the Y1 Fermi doublet. ThisFermi doublet is also sensitive to the hydrogen bonding
state of the tyrosine and the ratio of the intensity ofthe 852 cm�1 band to the 826 cm�1 band increases withincreasing enthalpy of hydrogen bond formation.19 – 22 Ourdata show a ratio of 0.97 for the complex and 0.98 foravidin alone, which suggests very little difference in thehydrogen bonding state of Tyr33 upon biotin binding, inagreement with previous visible and near infrared Ramanstudies.8 – 11 The x-ray structures of the holo and apoforms of avidin reveal that the ureido oxygen of biotinforms a hydrogen bond with the Tyr33 of holo avidin,with apo avidin having several water molecules in thebinding pocket, one being hydrogen bonded to Tyr33.23 – 25
This may account for the absence of a change in thehydrogen bonding state of Tyr33 indicated by our data.The displacement of water molecules from the bindingpocket and the formation of a hydrophobic and more polarenvironment upon biotin binding would account for all thechanges we observe in the UVRR bands of Tyr33.
Tryptophan
The dominant contributions to the difference spectrumcan be assigned to the tryptophan residues. The W3 peakwavenumber shows a strong correlation to the torsionalangle of tryptophan and the 1549 cm�1 peak in Fig. 1(a)and 1(b) represents an average value of approximately 90°
of j�2,1j for the four Trp residues in apo and holo avidin.The difference spectrum reveals that for holo avidin thereis an apparent increase in intensity and also splitting of theW3 peak, giving peaks at 1556 cm�1 and 1547 cm�1. Thesmall negative feature at 1585 cm�1 complicates the W3feature in the difference spectrum and is due to an upshiftin the W2 wavenumber profile. The increase in intensityof the W3 features is due to the expulsion of water andthe formation of a hydrophobic pocket with a concomitantdecrease in local dielectric constant when biotin is bound.The splitting of W3, revealed in the difference spectrum,reflects the reorganization of the tryptophan residues uponaddition of biotin.
The W7 Fermi doublet also shows a noticeable increasein intensity in the holo form. The ratio of the peak inten-sity of the 1359 cm�1 to the 1344 cm�1 band reflects thepolar nature of the Trp environment.12,26 Apo avidin has a1359/1344 cm�1 value of 1.08 and holo avidin 1.15. Theincrease in this ratio upon addition of biotin reflects theexpulsion of water and the hydrophobic nature of biotin.
All the other Trp features in the difference spectra at1492, 1462, 1432, 1238, 1009, 877 and 760 cm�1 aredue to increases in intensity from W4, W5, W6, W10,W16, W7 and W18, respectively. The general increasein intensity for the Trp UVRR bands upon addition ofbiotin is much more profoundly illustrated here than inprevious visible Raman studies and indicates a change toa more hydrophobic environment for these residues.8 – 11,16
The wavenumber of W17 is a marker of the degree ofhydrogen bonding of the Trp indole ring.27 No changein wavenumber is observed, only an increase in inten-sity, which indicates that there is no change in the aver-age degree of hydrogen bonding of the four avidin Trpresidues, similar to our observations concerning the hydro-gen bonding of Tyr33.
The UVRR difference spectrum presented here resolvesthe Tyr33 and the Trp contributions to the binding of biotinby avidin. Changes in the ultraviolet absorbance spectral
Copyright 2000 John Wiley & Sons, Ltd. J. Raman Spectrosc. 31, 373–375 (2000)
ULTRAVIOLET RESONANCE RAMAN STUDY OF THE AVIDIN BIOTIN COMPLEX 375
properties of Tyr33 are obscured by the dominant effectof Trp at pH 7.5.28 We have demonstrated the ability ofUVRR to give better molecular specificity, allowing forunambiguous assignment of the Raman peaks to molecularchanges to the Tyr33 residue of avidin. The UVRR methodalso allows changes in the molecular orientation and
environment of the Trp residues to be revealed in solutionmeasurements. Our results also indicate that there is littlechange in the hydrogen bonding state of Tyr33 or the Trpresidues and provide further evidence of the importanceof hydrophobic interactions in stabilizing the avidin/biotincomplex.
REFERENCES
1. Green NM. Adv. Protein Chem. 1975; 29: 85.2. Wilchek M, Bayer EA. Methods in Enzymology. Academic
Press: London, 1990.3. Gitlin G, Bayer EA, Wilchek M. Biochem. J. 1990; 269: 527.4. Gitlin G, Bayer EA, Wilchek M. Biochem. J. 1988; 250: 291.5. Mei G, Pugliese L, Rosato N, Toma L, Bolognesi M,
Finazziagro A. J. Mol. Biol. 1994; 242: 559.6. Kurzban GP, Gitlin G, Bayer EA, Wilchek M, Horowitz PM.
Biochemistry 1989; 28: 8537.7. Green NM. Biochem. J. 1963; 89: 599.8. Honzatko RB, Williams RW. Biochemistry 1982; 21: 6201.9. Fagnano C, Fini G, Torreggiani A. J. Raman Spectrosc. 1995;
26: 991.10. Fagnano C, Torreggiani A, Fini G. Biospectroscopy 1996; 2:
225.11. Torreggiani A, Fini G. J. Raman Spectrosc. 1998; 29: 229.12. Harada I, Takeuchi H. In Spectroscopy of Biological Systems,
Clark RJH, Hester RE (eds). Wiley: Wichester, 1986; 113 175.13. Williams KPJ, Pitt GD, Batchelder DN, Kip BJ. Appl.
Spectrosc. 1994; 48: 232.14. Hu XH, Spiro TG. Biochemistry 1997; 36: 15701.15. Kaminaka S, Kitagawa T. Appl. Spectrosc. 1992; 46: 1804.16. Hashimoto S, Sasaki H, Takeuchi H. J. Am. Chem. Soc. 1998;
120: 443.
17. Ames JB, Ros M, Raap J, Lugtenburg J, Mathies RA.Biochemistry 1992; 31: 5328.
18. Hashimoto S, Takeuchi H, Nakagawa M, Tsuda M. FebsLetters 1996; 398: 239.
19. Rodgers KR, Su C, Subramaniam S, Spiro TG. J. Am. Chem.Soc. 1992; 114: 3697.
20. Hildebrandt PG, Copeland RA, Spiro TG, Otlewski J,Laskowski M, Prendergast FG. Biochemistry 1988; 27:5426.
21. Tu AT. In Spectroscopy of Biological Systems, Clark RJH,Hester RE (eds). Wiley: Chichester, 1986; 47 112.
22. Couling VW, Foster NW, Klenerman D. J. of RamanSpectrosc. 1997; 28: 33.
23. Pugliese L, Coda A, Malcovati M, Bolognesi M. J. Mol. Biol.1993; 231: 698.
24. Pugliese L, Malcovati M, Coda A, Bolognesi M. J. Mol. Biol.1994; 235: 42.
25. Livnah O, Bayer EA, Wilchek M, Sussman JL. Proc. Natl.Acad. Sci. U.S.A. 1993; 90: 5076.
26. Harada I, Miura T, Takeuchi H. Spectrochimica Acta Part A1986; 42: 307.
27. Miura T, Takeuchi H, Harada I. Biochemistry 1988; 27: 88.28. Green NM. Biochem. J. 1963; 89: 609.
Copyright 2000 John Wiley & Sons, Ltd. J. Raman Spectrosc. 31, 373–375 (2000)
