Ultraviolet Resonance Raman Studies of Quaternary Structure of Hemoglobin Using a Tryptophan β37 Mutant

Nagai, Masako; Kaminaka, Shoji; Ohba, Yusuke; Nagai, Yukifumi; Mizutani, Yasuhisa; Kitagawa, Teizo

doi:10.1074/jbc.270.4.1636

“…44 interface, its UVRR intensity is particularly sensitive to the quaternary structure. 33 The W17 band is sensitive to hydrogen bonding of the NH part of the indole side chain. 1 In fact, the W17 frequency of a-lactalbumin exhibits an upshift upon exposure of Trp to the solvent.…”

Section: Tryptophan-related Modesmentioning

confidence: 99%

Raman Spectroscopy of Proteins

Kitagawa¹,

Hirota²

2001

Handbook of Vibrational Spectroscopy

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The sections in this article are Introduction General Aspects of Ordinary R aman Spectra of Proteins Amides I and III Frequencies and Secondary Structures T rp Bands and Microenvironments of T rp Residues T yr Bands and Hydrogen Bonding Histidine Band and Protonation S  S Stretching Frequency and Conformation of Disulfide Bridge UV Resonance R aman Spectra of Proteins Amide Modes Tyrosine‐Related Modes Tryptophan‐Related Modes Histidine‐Related Modes Cysteine‐Related Modes X ‐Proline Vibrational Modes Modes of Metal‐Coordinated Residues Visible Resonance R aman Spectra of Proteins Heme Proteins Heme Skeletal Vibrational Modes Fe  H is and Fe  C ys stretching modes Fe ( II )‐ O 2 Related Modes Fe ( II )‐ CO Related Modes Fe ( III )‐ CN Related Modes Fe ( II )‐ NO and Fe ( III )‐ NO Related Modes Fe ( III )‐ OH Related Modes Fe ( IV ) O Related Modes Copper Proteins Quinone Vibrations of Quinoproteins Tyrosine Radical Modes in Proteins Flavo Proteins Vis RR Spectra of Other Proteins

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“…The deoxy-HbA minus CO-HbA solution phase UVRR difference spectrum (Fig. 1b) is primarily reflective of changes in the R-T sensitive ␣ 1 ␤ 2 dimer interface arising from differences between the deoxy T and liganded R conformations (7,(23)(24)(25)(26)(27)(28)(29). The W3 difference feature at 1549 cm Ϫ1 (Fig.…”

Section: Ligand Binding To Sol-gel Encapsulated Hemoglobin 30358mentioning

confidence: 99%

“…1b) on the low frequency side of the central peak at 1559 cm Ϫ1 (Fig. 1a) originates from a T-R conformational difference in Trp␤37, the pivotal residue in the hinge region of the ␣ 1 ␤ 2 interface (7,23,26,28). Whereas Trp␤37 is responsible for the low frequency shoulder of the W3 band, Trp␣14 and Trp␤15, located on the A helices, are the source of the central W3 peak at 1559 cm Ϫ1 (7,21).…”

Section: Ligand Binding To Sol-gel Encapsulated Hemoglobin 30358mentioning

confidence: 99%

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

Juszczak

¹

,

Friedman

²

1999

Journal of Biological Chemistry

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We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxyHbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an ␣14␤15 Trp W3 band difference at 1559 cm ؊1 . The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm ؊1 ) UVRR difference bands suggest conformational shifts for the penultimate Tyr␣140 on the F helix, the "switch" region Tyr␣42, and the "hinge" region Trp␤37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.A molecular level understanding of how proteins function requires not only a three-dimensional picture of equilibrium structures but also a time-ordered sequence of functionally related molecular events. The second requirement is difficult because of the challenge of either trapping detectable populations of nonequilibrium structures or creating a synchronized nonequilibrium population whose dynamics can be followed.Because protein dynamics can span many decades in time starting on the picosecond time scale, it is often necessary to prepare the evolving population using a short, laser-derived excitation pulse. This approach has been very useful when starting with ligand-saturated hemeproteins. Nanosecond and faster photodissociation of ligand-bound hemoglobins has shed considerable light on the sequence of molecular steps associated with the tertiary and quaternary structure changes in the globin that follow ligand dissociation (2-10).The sequence of conformational events following solventderived (non-geminate) ligand binding to deoxy hemoglobin is much more of a challenge because of the temporal constraints imposed by ligand diffusion. The initial conformational responses to ligand or substrate binding to a given protein will typically be faster than the diffusion time that is inherent to any rapid mixing experiments. In the present work, an approach to overcoming this limitation, based on sol-gel encapsulation, is presented in which the conformational response time is extended well beyond the ligand diffusion time. Thus we have been able to use UVRR 1 to probe the initial sequence of conformation changes in hemoglobin when CO binds to the equilibrium form of deoxy-HbA.The encapsulation of proteins in porous TMOS-derived solgels has been shown to oc...

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“…Studies of Hb Hirose ( 37Trp f Ser) (16) and Hb Rothchild ( 37Trp f Arg) (14) have indicated that the change of Trp UVRR bands was ascribed to 37Trp. n the other hand, the spectral changes of Tyr UVRR bands were interpreted to arise from hydrogen bond formation between R42Tyr and 99Asp upon the R-T quaternary transition (14).…”

mentioning

confidence: 97%

Quaternary Structure Sensitive Tyrosine Residues in Human Hemoglobin: UV Resonance Raman Studies of Mutants at α140, β35, and β145 Tyrosine

Nagai

¹

,

Wajcman

²

,

Lahary

³

et al. 1999

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Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.

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Ultraviolet Resonance Raman Studies of Quaternary Structure of Hemoglobin Using a Tryptophan β37 Mutant

Cited by 53 publications

References 28 publications

Raman Spectroscopy of Proteins

Raman Spectroscopy of Proteins

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

Quaternary Structure Sensitive Tyrosine Residues in Human Hemoglobin: UV Resonance Raman Studies of Mutants at α140, β35, and β145 Tyrosine

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