Subnanosecond motions of tryptophan residues in proteins.
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Journal: 1979/May - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 284374
Abstract:
The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.
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Proc Natl Acad Sci U S A 76(1): 56-60
PMID: 284374

Subnanosecond motions of tryptophan residues in proteins

Abstract

The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (ϕ) of 9.9 nsec at 20°C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits ϕ of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8°C (ϕ = 31.4 nsec), whereas at 43°C it rotates rapidly (ϕ1 = 0.14 nsec) within a cone of semiangle 26° in addition to rotating together with the whole protein (ϕ2 = 14 nsec). Of particular interest in the large angular range (semiangle, 34°) and fast rate (ϕ1 = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.

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Selected References

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  • Hammes GG, Wu CW. Kinetics of allosteric enzymes. Annu Rev Biophys Bioeng. 1974;3(0):1–33. [PubMed] [Google Scholar]
  • Weber G. Ligand binding and internal equilibria in proteins. Biochemistry. 1972 Feb 29;11(5):864–878. [PubMed] [Google Scholar]
  • Lakowicz JR, Weber G. Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry. 1973 Oct 9;12(21):4171–4179. [PubMed] [Google Scholar]
  • McCammon JA, Gelin BR, Karplus M. Dynamics of folded proteins. Nature. 1977 Jun 16;267(5612):585–590. [PubMed] [Google Scholar]
  • Yguerabide J, Epstein HF, Stryer L. Segmental flexibility in an antibody molecule. J Mol Biol. 1970 Aug;51(3):573–590. [PubMed] [Google Scholar]
  • Lovejoy C, Holowka DA, Cathou RE. Nanosecond fluorescence spectroscopy of pyrenebutyrate anti-pyrene antibody complexes. Biochemistry. 1977 Aug 9;16(16):3668–3672. [PubMed] [Google Scholar]
  • Mendelson RA, Morales MF, Botts J. Segmental flexibility of the S-1 moiety of myosin. Biochemistry. 1973 Jun 5;12(12):2250–2255. [PubMed] [Google Scholar]
  • Brown JR. Structural origins of mammalian albumin. Fed Proc. 1976 Aug;35(10):2141–2144. [PubMed] [Google Scholar]
  • Davis A, Moore IB, Parker DS, Taniuchi H. Nuclease B. A possible precursor of nuclease A, an extracellular nuclease of Staphylococcus aureus. J Biol Chem. 1977 Sep 25;252(18):6544–6553. [PubMed] [Google Scholar]
  • Eylar EH, Brostoff S, Hashim G, Caccam J, Burnett P. Basic A1 protein of the myelin membrane. The complete amino acid sequence. J Biol Chem. 1971 Sep 25;246(18):5770–5784. [PubMed] [Google Scholar]
  • Yguerabide J. Nanosecond fluorescence spectroscopy of macromolecules. Methods Enzymol. 1972;26:498–578. [PubMed] [Google Scholar]
  • Belford GG, Belford RL, Weber G. Dynamics of fluorescence polarization in macromolecules. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1392–1393.[PMC free article] [PubMed] [Google Scholar]
  • Kinosita K, Jr, Kawato S, Ikegami A. A theory of fluorescence polarization decay in membranes. Biophys J. 1977 Dec;20(3):289–305.[PMC free article] [PubMed] [Google Scholar]
  • Grinvald A, Steinberg IZ. On the analysis of fluorescence decay kinetics by the method of least-squares. Anal Biochem. 1974 Jun;59(2):583–598. [PubMed] [Google Scholar]
  • Grinvald A. The use of standards in the analysis of fluorescence decay data. Anal Biochem. 1976 Sep;75(1):260–280. [PubMed] [Google Scholar]
  • Veatch WR, Stryer L. Effect of cholesterol on the rotational mobility of diphenylhexatriene in liposomes: a nanosecond fluorescence anisotrophy study. J Mol Biol. 1977 Dec 25;117(4):1109–1113. [PubMed] [Google Scholar]
  • Rosen P, Pecht I. Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa. Biochemistry. 1976 Feb 24;15(4):775–786. [PubMed] [Google Scholar]
  • Grinvald A, Schlessinger J, Pecht I, Steinberg IZ. Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization. Biochemistry. 1975 May 6;14(9):1921–1929. [PubMed] [Google Scholar]
  • Hirshfeld H, Teitelbaum D, Arnon R, Sela M. Basic encephalitogenic protein: A simplified purification on sulphoethyl-sephadex. FEBS Lett. 1970 May 1;7(4):317–320. [PubMed] [Google Scholar]
  • WEBER G. Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan and related compounds. Biochem J. 1960 May;75:335–345.[PMC free article] [PubMed] [Google Scholar]
  • WEBER G. Fluorescence-polarization spectrum and electronic-energy transfer in proteins. Biochem J. 1960 May;75:345–352.[PMC free article] [PubMed] [Google Scholar]
  • Cotton FA, Bier CJ, Day VW, Hazen EE, Jr, Larsen S. Some aspects of the structure of staphylococcal nuclease. I. Crystallographic studies. Cold Spring Harb Symp Quant Biol. 1972;36:243–249. [PubMed] [Google Scholar]
  • Eylar EH, Thompson M. Allergic encephalomyelitis: the physico-chemical properities of the basic protein encephalitogen from bovine spinal cord. Arch Biochem Biophys. 1969 Feb;129(2):468–479. [PubMed] [Google Scholar]
  • Krigbaum WR, Hsu TS. Molecular conformation of bovine A1 basic protein, a coiling macromolecule in aqueous solution. Biochemistry. 1975 Jun 3;14(11):2542–2546. [PubMed] [Google Scholar]
  • Eylar EH, Caccam J, Jackson JJ, Westall FC, Robinson AB. Experimental allergic encephalomyelitis: synthesis of disease-inducing site of the basic protein. Science. 1970 Jun 5;168(3936):1220–1223. [PubMed] [Google Scholar]
  • Finazzi-Agrò A, Rotilio G, Avigliano L, Guerrieri P, Boffi V, Mondovì B. Environment of copper in Pseudomonas fluorescens azurin: fluorometric approach. Biochemistry. 1970 Apr 28;9(9):2009–2014. [PubMed] [Google Scholar]
  • Adman ET, Stenkamp RE, Sieker LC, Jensen LH. A crystallographic model for azurin a 3 A resolution. J Mol Biol. 1978 Jul 25;123(1):35–47. [PubMed] [Google Scholar]
  • Visscher RB, Gurd FR. Rotational motions in myoglobin assessed by carbon 13 relaxation measurements at two magnetic field strengths. J Biol Chem. 1975 Mar 25;250(6):2238–2242. [PubMed] [Google Scholar]
  • Wüthrich K, Wagner G. NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor. FEBS Lett. 1975 Feb 1;50(2):265–268. [PubMed] [Google Scholar]
  • Hull WE, Sykes BD. Fluorotyrosine alkaline phosphatase: internal mobility of individual tyrosines and the role of chemical shift anisotropy as a 19F nuclear spin relaxation mechanism in proteins. J Mol Biol. 1975 Oct 15;98(1):121–153. [PubMed] [Google Scholar]
  • Snyder GH, Rowan R, 3rd, Karplus S, Sykes BD. Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor. Biochemistry. 1975 Aug 26;14(17):3765–3777. [PubMed] [Google Scholar]
  • Hetzel R, Wüthrich K, Deisenhofer J, Huber R. Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). II. Semi-empirical energy calculations. Biophys Struct Mech. 1976 Aug 23;2(2):159–180. [PubMed] [Google Scholar]
Department of Structural Biology, Sherman Fairchild Center, Stanford University School of Medicine, Stanford, California 94305
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Abstract
The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (ϕ) of 9.9 nsec at 20°C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits ϕ of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8°C (ϕ = 31.4 nsec), whereas at 43°C it rotates rapidly (ϕ1 = 0.14 nsec) within a cone of semiangle 26° in addition to rotating together with the whole protein (ϕ2 = 14 nsec). Of particular interest in the large angular range (semiangle, 34°) and fast rate (ϕ1 = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.
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