Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Loewenthal, Ron; Sancho, Javier; Fersht, Alan R.

doi:10.1021/bi00241a021

Cited by 139 publications

(109 citation statements)

References 23 publications

(20 reference statements)

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…The concentration of protein in the solution was determined spectrophotometrically at 280 nm using the extinction coefficient Eii:;Lvo = 22.1, measured in our laboratory from the nitrogen content using the method described by Jaenicke (1974). The same coefficient has been obtained before by Loewenthal et al (1991), who have calculated it from the amino acid composition of barnase using the method of Gill and von Hippel (1989). This value is close to the extinction coefficient of 21 .O reported by Hartley (1975).…”

Section: Methodssupporting

confidence: 85%

Thermodynamics of barnase unfolding

et al. 1994

View full text Add to dashboard Cite

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 "C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features -the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.Keywords: barnase; hydrogen bonding; hydrophobic interactions; scanning microcalorimetry Barnase attracts the special interest of researchers engaged in studying protein folding because it is one of the smallest globular proteins (1 10 amino acid residues; see Kinemage 1) that does not have disulfide crosslinks but is quite stable and unfolds reversibly. The latter circumstance is very important for quantitative analysis of this process by equilibrium thermodynamics, specifying the stability of the native state in energetic terms.In solutions close to neutral pH, reversible unfolding of barnase by denaturants and temperature appears to be a 2-state transition (Hartley, 1968(Hartley, , 1975Makarov et al., 1993). Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition. However, later in 1993 the paper of Makarov et al. (1993) ing subdomains. Analysis of the 3-dimensional structure of these proteins does indeed reveal 2 hydrophobic clusters (see Kinemage 1;Serrano et al., 1992). Therefore, the mode of barnase denaturation required detailed investigation. Because there was also some deviation between our estimates of the enthalpy of denaturation and that of Makarov et al. (1993), we felt it necessary to repeat calorimetric measurements on barnase to ...

show abstract

Section: Methodssupporting

confidence: 85%

Thermodynamics of barnase unfolding

et al. 1994

View full text Add to dashboard Cite

show abstract

“…In the model the loop formed in the N-terminal tail brings His ]6 into line with the axis of the A helix and Arg ~5 close to His 16 and His 19 (which is at the N-terminus of helix A). The influence of the positive charges of the helix dipole and the arginine side chain would be expected to lower the pK, of these histidine residues [28,30], as was observed. His 71 shows two independent titrations, the first reflecting a carboxyl titration (pK,2.6) and the second representing the imidazolium pKa, which is slightly elevated at 7.4.…”

Section: Discussionmentioning

confidence: 75%

“…As the pK~ values were not corrected for isotope effects, values in H20 may be slightly lower [28]. The histidine residues were sequentially assigned by identifying the missing imidazole proton resonances in 1D and 2D TOCSY spectra of each of the five His ~ Ala mutants.…”

Section: Pk a Valuesmentioning

confidence: 99%

Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor

et al. 1994

View full text Add to dashboard Cite

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with ~H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pKa values of which range from 3.6 to 7.4. These pKa values were assigned to individual histidine residues from NMR studies on a series of His --* Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.

show abstract

“…1) to wild-type B domain (with rms deviation of 1.3 Å) (12). Trp-15 lies on the surface of H1 and interacts with the side chain of His-19, which quenches its fluorescence in the native structure, as found for the His-18͞ Trp-94 interaction in barnase (13) (Fig. 2).…”

Section: Structural and Kinetic Characterization Of Mutantsmentioning

confidence: 78%

“…The genes for mutants were prepared by using QuikChange (Stratagene), and the proteins were produced as described above. 13 C-and 15 N-labeled protein was prepared by using the K-Mops minimal media. All proteins were Ͼ95% pure.…”

Section: Methodsmentioning

confidence: 99%

Testing protein-folding simulations by experiment: B domain of protein A

Sato

Religa

Daggett

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

135

262

View full text Add to dashboard Cite

We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental ⌽ values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable ␣-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation-condensation. None of the published atomistic simulations are fully consistent with the experimental picture although many capture important features. There is a continuing need for combining simulation with experiment to describe folding pathways, and of continued testing to improve predictive methods.

show abstract

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Cited by 139 publications

References 23 publications

Thermodynamics of barnase unfolding

Thermodynamics of barnase unfolding

Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor

Testing protein-folding simulations by experiment: B domain of protein A

Contact Info

Product

Resources

About

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Cited by 139 publications

References 23 publications

Thermodynamics of barnase unfolding

Thermodynamics of barnase unfolding

Homology modelling and 1H NMR studies of human leukaemia inhibitory factor

Testing protein-folding simulations by experiment: B domain of protein A

Contact Info

Product

Resources

About

Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor