Skip to main content
SpringerLink
Log in

Compact state of a protein molecule with pronounced small-scale mobility: bovine α-lactalbumin

  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine α-lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine α-lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.

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

Similar content being viewed by others

Abbreviations

Bα-LA:

bovine α-lactalbumin

Gu·HCl:

guanidine hydrochloride

CD:

circular dichroism

UV:

ultraviolet

IR:

infra-red

NMR:

nuclear magnetic resonance. Differen forms of Bα-LA are abbreviated as follows

N:

native form

A:

acid form

T:

temperature-denatured form

U:

unfolded form (by 6 M Gu·HCl or 8 M urea). All forms have intact S-S bonds

References

  • Abaturov LV, Yakobashvili NN, Jinoria KSh, Molchanova TP, Varshavsky YaM (1976) Effect of inter-subunit contact on intramolecular conformational motility (conformational stability) of hemoglobin as revcaled by hydrogen exchange. FEBS Lett 70:127–130

    Google Scholar 

  • Ananthanarayanan VS, Ahmad F (1977) Evidence from rotatory measurement for an intermediate state in the guanidine hydrochloride denaturation of β-lactoglobulin. Can J Biochem 55:239–243

    Google Scholar 

  • Armstrong JMcD, McKenzie HA, Swayer WH (1967) On the fractionation of β-lactalbumin and α-lactalbumin. Biochim Biophys Acta 147:60–72

    Google Scholar 

  • Armstrong JMcD, Hpper KE, McKenzie HA, Murphy WH (1970) On the column chromatography of bovine whey proteins. Biochim Biophys Acta 214:419–426

    Google Scholar 

  • Brew K, Castelino FJ, Vanaman TC, Hill RI (1970) The complete amino acid sequence of bovine α-lactalbumin. J Biol Chem 245:4570–4582

    Google Scholar 

  • Chirgadze YuN, Shestopalov BV, Venyaminov SYu (1973) Intensities and other spectral parameters of infrared amide bands of polypeptides in the β- and random forms. Biopolymers 12:1337–1351

    Google Scholar 

  • Chirgadze YuN, Brazhnikov EV (1974) Intensities and other spectral parameters of infrared amide bands of polypeptides in the α-helical form. Biopolymers 13:1701–1712

    Google Scholar 

  • Chirgadze YuN, Fedorov OV, Trushina NP (1975) Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water. Biopolymers 14:679–694

    Google Scholar 

  • Creighton TE (1980) Kinetic study of protein unfolding and refolding using urea gradient electrophoresis. J Mol Biol 137:61–80

    Google Scholar 

  • Creighton TE, Pain RH (1980) Unfolding and refolding of Staphylococcus aureus penicillinase by urea-gradient electrophoresis. J Mol Biol 137:431–436

    Google Scholar 

  • Denton JB, Konishi Y, Scherega HA (1982) Folding of ribonuclease A from a partially disordered conformation. Kinetic study under folding conditions. Biochemistry 21: 5155–5163

    Google Scholar 

  • Dolgikh DA, Gilmanshin RI, Brazhnikov EV, Bychkova VE, Semisotnov GV, Venyaminov SYu, Ptitsyn OB (1981) α-Lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett 136:311–315

    Google Scholar 

  • Dolgikh DA, Abaturov LV, Brazhnikov EV, Lebedev YuO, Chirgadze YuN, Ptitsyn OB (1983) Acid form of carbonic anhydrase: “molten globule” with a secondary structure. Dokl Akad Nauk SSSR 272:1481–1484

    Google Scholar 

  • Dolgikh DA, Kolomiets AP, Bolotina IA, Ptitsyn OB (1984) “Molten globule” state accumulates in carbonic anhydrase folding. FEBS Lett 165:88–92

    Google Scholar 

  • Englander SW, Downer N, Teitelbaum H (1972) Hydrogen exchange. Ann Rev Biochem 41:903–924

    Google Scholar 

  • Guinier A, Fournet G (1955) Small-angle scattering of X-rays. J. Wiley & sons, New York; Chapman & Hall, London

    Google Scholar 

  • Hiraoka Y, Segawa T, Kuwajima K, Sugai S, Murai N (1980) α-Lactalbumin: a calcium metalloprotein. Biochem Biophys Res Commun 95:1098–1104

    Google Scholar 

  • Holladay LA, Hammonds RG, Puett D (1974) Growth hormone conformation and conformational equilibria. Biochemistry 13:1653–1661

    Google Scholar 

  • Kato S, Okamura M, Shimamoto N, Utiyama H (1981) Spectral evidence for a rapidly formed structural intermediate in the refolding kinetics of hen egg-white lysozyme. Biochemistry 20:1080–1085

    Google Scholar 

  • Kim PS, Baldwin RL (1980) Structural intermediates trapped during the folding of ribonuclease A by amide proton exchange. Biochemistry 19:6124–6129

    Google Scholar 

  • Kim PS, Baldwin RL (1982) Specific intermediates in the folding reactions of small proteins and the mechanisms of protein folding. Ann Rev Biochem 51:459–489

    Google Scholar 

  • Kita N, Kuwajima K, Nitta K, Sugai S (1976) Equilibrium and kinetics of the unfolding of α-lactalbumin by guanidine hydrochloride (II). Biochim Biophys Acta 427: 350–358

    Google Scholar 

  • Krigbaum WR, Kügler FR (1970) Molecular conformation of egg-white lysozyme and bovine α-lactalbumin in solution. Biochemistry 9:1216–1223

    Google Scholar 

  • Kronman MJ, Andreotti R, Vitols R (1964) Inter- and intramolecular interactions of α-lactalbumin. II. Aggregation reactions at acid pH. Biochemistry 3:1152–1160

    Google Scholar 

  • Kronman MJ, Cerankowski L, Holmes LG (1965) Inter- and intramolecular interactions of α-lactalbumin. III. Spectral changes at acid pH. Biochemistry 4:518–525

    Google Scholar 

  • Kronman MJ, Holmes LG (1965) Inter- and intramolecular interactions of α-lactalbumin. IV. Location of tryptophan groups. Biochemistry 4:526–532

    Google Scholar 

  • Kroman MJ, Blum R, Holmes LG (1966) Inter- and intramolecular interactions of α-lactalbumin. VI. Optical rotation dispersion properties. Biochemistry 5:1970–1978

    Google Scholar 

  • Kronman MJ, Holmes LG, Robbins EM (1967) Inter- and intramolecular interactions of α-lactalbumin. VIII. The alkaline conformational change. Biochim Biophys Acta 133:46–55

    Google Scholar 

  • Kronman MJ, Holmes LG, Robbins EM (1971) Enter- and intramolecular interactions of α-lactalbumin. X. Effect of acylation of tyrosyl and lysyl side chains on molecular conformations. J Biol Chem 246:1909–1921

    Google Scholar 

  • Kronman MJ, Sinha KS, Brew K (1981) Characteristics of the binding of Ca2+ and other divalent metal ions to bovine α-lactalbumin. J Biol Chem 256:8582–8587

    Google Scholar 

  • Kuwajima K, Nitta K, Sugai S (1975) Electrophoretic investigations of the acid conformational change of α-lactalbumin. J Biochem (Tokyo) 78:205–211

    Google Scholar 

  • Kuwajima K, Nitta K, Yoneyama M, Sugai S (1976) Threestate denaturation of α-lactalbumin by guanidine hydrochloride. J Mol Biol 106:359–373

    Google Scholar 

  • Kuwajima K (1977) A folding model of α-lactalbumin deduced from the three-state denaturation mechanism. J Mol Biol 114:241–258

    Google Scholar 

  • Kuwajima K, Sugai S (1978) Equilibrium and kinetics of the thermal unfolding of α-lactalbumin. The relation of its folding mechanism. Biophys Chem 8:247–254

    Google Scholar 

  • Kuwajima K, Ogawa Y, Sugai S (1981) Role of the interaction between ionizable groups in the folding of bovine α-lactalbumin. J Biochem (Tokyo) 89:759–770

    Google Scholar 

  • Lebedev YuO, Abaturov LV, Pirtskhalava MK, Varshavsky YaM (1976) Hydrogen-exchange study in ribonuclease A. Stud Biophys 60:143–148

    Google Scholar 

  • Lifshitz IM, Grossberg AYu, Khokhlov AR (1979) Volume interactions in the statistical physics of a polymer macromolecule. Usp Fiz Nauk (USSR) 127:353–389

    Google Scholar 

  • Maruyama S, Kuwajima K, Nitta K, Sugai S (1977) Thermodynamic characterization of partially denatured states in the denaturation process of bovine α-lactalbumin by inorganic denaturants. Biochim Biophys Acta 494:343–353

    Google Scholar 

  • McCoy LF, Rowe ES, Wong K-P (1980) Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B. Biochemistry 19:4738–4743

    Google Scholar 

  • Molday RS, Englander SW, Kallen RG (1972) Primary structure effects on peptide group hydrogen exchange. Biochemistry 11:150–158

    Google Scholar 

  • Nozaka M, Kuwajima K, Nitta K, Sugai S (1978) Detection and characterization of the intermediate on the folding pathway of human α-lactalbumin. Biochemistry 17: 3753–3758

    Google Scholar 

  • Nozaki Y (1972) The preparation of guanidine hydrochloride. In: Hirs CH, Timasheff SN (eds) Methods in Enzymology, Vol 26, Academic Press, New York London, pp 43–50

    Google Scholar 

  • Ohgushi M, Wada A (1983) “Molten-globule state”: a compact form of globular proteins with mobile side-chains. FEBS Lett 164:21–24

    Google Scholar 

  • Permyakov EA, Kalinichenko LP, Morozova LA, Yarmolenko VV, Burstein EA (1981 a) α-Lactalbumin binds magnesium ions: study by means of intrinsic fluorescence technique. Biochem Biophys Res Commun 102:1–7

    Google Scholar 

  • Permyakov EA, Yarmolenko VV, Kalinichenko LP, Morozova LA, Burstein EA (1981 b) Calcium binding to α-lactalbumin: structural rearrangement and association constant evaluation by means of intrinsic protein fluorescence changes. Biochem Biophys Res Commun 100:191–197

    Google Scholar 

  • Pessen H, Humosinski TF, Timasheff SN (1971) The use of small-angle X-ray scattering to determine protein conformation. J Agricult Food Chem 19:698–702

    Google Scholar 

  • Pfeil W (1981) Thermodynamics of α-lactalbumin unfolding. Biophys Chem 13:181–186

    Google Scholar 

  • Privalov PL (1976) Thermodynamic investigations of biological macromolecules. Pure Appl Chem 47:293–304

    Google Scholar 

  • Privalov PL (1979) Stability of proteins. Small globular proteins. Adv Protein Chem 33:167–241

    Google Scholar 

  • Privalov PL, Plotnikov VV, Filimonov VV (1975) Precision scanning calorimeter for the study of liquids. J Chem Thermodyn 7:41–47

    Google Scholar 

  • Provencher SW, Glöckner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33–37

    Google Scholar 

  • Ptitsyn OB (1973) Step-wise mechanism of self-organization of protein molecules. Dokl Akad Nauk SSSR 210: 1213–1215

    Google Scholar 

  • Ptitsyn OB, Eizner YuE (1965) Theory of globule-coil transitions in macromolecules. Biofizika (USSR) 10:3–6

    Google Scholar 

  • Robbins EM, Holmes LG (1970) Circular dichroism spectra of α-lactalbumin. Biochim Biophys Acta 221:234–240

    Google Scholar 

  • Schmid FX, Baldwin RL (1979) Detection of an early intermediate in the folding of ribonuclease A by protection of amide protons against exchange. J Mol Biol 135:199–215

    Google Scholar 

  • Schmid FX, Blaschek H (1981) A native-like intermediate on the ribonuclease A folding pathway. 2. Comparison of its properties to native ribonuclease A. Eur J Biochem 114: 111–117

    Google Scholar 

  • Schulz GE (1979) Nucleotide binding proteins. In: Balaban M (ed) Molecular mechanisms of biological recognition. Elsevier/North-Holland Biomed. Press, Amsterdam Oxford New York, pp 79–94

    Google Scholar 

  • Sears DW, Beychok S (1973) Circular dichroism. In: Leach SJ (ed) Physical principles and techniques of protein chemistry, part C. Academic Press, New York London, pp 445–593

    Google Scholar 

  • Segawa T, Sugai S (1983) Interactions of divalent metal ions with bovine, human and goat α-lactalbumins. J Biochem (Tokyo) 93:1321–1328

    Google Scholar 

  • Semisotnov GV, Anufrieva EV, Zikherman KKh, Kasatkin SB, Ptitsyn OB (1981) Polarized luminescence and mobility of tryptophan residues in polypeptide chains. Biopolymers 20:2287–2309

    Google Scholar 

  • Serdyuk IN, Grenader AK (1975) Joint use of light, X-ray and neutron scattering for investigation of RNA and protein mutual distribution within the 50S subparticle of E. coli ribosomes. FEBS Lett 59:133–136

    Google Scholar 

  • Shakhnovich EI, Finkelstein AV (1982) On the theory of cooperative transitions in protein globules. Dokl Akad Nauk SSSR 267:1247–1250

    Google Scholar 

  • Schchedrin BM, Feigin LA (1966) Calculation of collimation correction in small-angle X-ray scattering. Finite dimensions of slit. Kristallografiya (USSR) 11:159–163

    Google Scholar 

  • Sommers PB, Kronman MJ (1980) Comparative fluorescence properties of bovine, goat, human and guinea pig α-lactalbumin. Characterization of the environments of individual tryptophan residues in partially folded conformers. Biophys Chem 11:217–232

    Google Scholar 

  • Takesada H, Nakanishi M, Tsuboi M (1973) Structure of α-lactalbumin and its fluctuation. J Mol Biol 77:605–614

    Google Scholar 

  • Tanford Ch (1968) Protein denaturation. Adv Protein Chem 23:121–282

    Google Scholar 

  • Vainstein BK, Feigin LA (1965) Ordered arrangement of molecules of soluble ribonucleic acid in water solutions. Dokl Akad Nauk SSSR 161:1444–1447

    Google Scholar 

  • Weber G (1952) Polarization of the fluorescence of macromolecules. Biochem J 51:145–155

    Google Scholar 

  • Wetlaufer DB (1961) Osmometry and general characterization of α-lactoglobulin. C R Trav Lab Carlsberg 32:125–138

    Google Scholar 

  • Wong K-P, Hamlin LM (1974) Acid denaturation of bovine carbonic anhydrase B. Biochemistry 13:2678–2683

    Google Scholar 

  • Wong K-P, Tanford Ch (1973) Denaturation of bovine carbonic anhydrase B by guanidine hydrochloride. A process involving separable sequental conformational transitions. J Biol Chem 248:8518–8523

    Google Scholar 

  • Woodward CR, Rosenberg A (1970) Oxidized rNase as a protein model having no contribution to the hydrogen exchange rate from conformational restrictions. Proc Natl Acad Sci USA 66:1067–1074

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Protein Research, Academy of Sciences of the USSR, SU-142292, Pushchino, Moscow Region, USSR

    D. A. Dolgikh, E. V. Brazhnikov, V. E. Bychkova, R. I. Gilmanshin, G. V. Semisotnov & O. B. Ptitsyn

  2. Institute of Molecular Biology, Academy of Sciences of the USSR, SU-117312, Moscow, USSR

    D. A. Dolgikh, L. V. Abaturov, I. A. Bolotina, E. V. Brazhnikov, V. E. Bychkova, R. I. Gilmanshin, Yu. O. Lebedev, G. V. Semisotnov & E. I. Tiktopulo

  3. Institute of Biophysics, Academy of Sciences of the USSR, SU-142292, Pushchino, Moscow Region, USSR

    L. V. Abaturov, I. A. Bolotina, Yu. O. Lebedev, E. I. Tiktopulo & O. B. Ptitsyn

Authors
  1. D. A. Dolgikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. V. Abaturov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. I. A. Bolotina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. V. Brazhnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. E. Bychkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R. I. Gilmanshin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yu. O. Lebedev
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. G. V. Semisotnov
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. E. I. Tiktopulo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. O. B. Ptitsyn
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dolgikh, D.A., Abaturov, L.V., Bolotina, I.A. et al. Compact state of a protein molecule with pronounced small-scale mobility: bovine α-lactalbumin. Eur Biophys J 13, 109–121 (1985). https://doi.org/10.1007/BF00256531

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00256531

Key words

Search

Navigation