Topics:

  -What do we measure with UVRR?
  -What did UVRR tell us about Hb A and Hb S tetramers?
  -How do Hb S fibers compare with Hb S tetramers?
  -Other structural changes observed

What do we measure with UVRR?

Previously, we have used Ultraviolet Resonance Raman (UVRR) spectroscopy to look at individual Hb S molecules and Hb S fibers.  Hemoglobin, itself, has been well characterized using UVRR spectroscopy by the Spiro  and Friedman  labs.  These labs have demonstrated that important hydrogen (H)-bonds formed across the a₁b₂ interface that stabilize the T- or deoxy state are observable by UVRR spectroscopy.  These important H-bonds are formed between the b37 Trp and a94 Asp residues and the a42 Tyr and b99 Asp residues (FIG 9.1).  In the UVRR spectrum, we observe signals associated with Trp and Tyr that either increase in intensity or shift in frequency because of H-bond formation.

What did UVRR tell us about Hb A and Hb S tetramers?

Our initial studies with UVRR spectroscopy (link to pdf of published paper 105 kb) showed that the H-bonds formed in Hb S were the same as those formed in Hb A.  (FIG 9.2)  From this we could conclude that the overall structure or the quaternary structure of Hb S in the T-state was the same as Hb A.  However, the UVRR spectra also indicated that there were subtle differences between the two proteins.  In particular, these spectra demonstrated that the environment of Trp residues in Hb S were slightly different from those in Hb A.  Specifically, from the frequency of the peak we could determine that either the a14 or b15 Trp residue was in a more hydrophobic environment in Hb S relative to Hb A.  This was true in both the oxy and the deoxy states (FIG 9.3).  Since the b15 Trp residue is close to the site of mutation (b6Glu®Val), we attribute the changes observed to that residue.  In terms of the protein, the different environment for the Trp residue suggested that the helix that it is located on, the a-helix, is closer to the interior of the protein in Hb S relative to Hb A.

FIG 9.2 How does FmetHb S compare to FmetHb A? Quaternary state contacts are the same for Hb A and Hb S Intersubunit H-bonds characteristic of the T-state are observed  Sokolov and Mukerji, J. Phys, Chem. B (1998) 102, 8314-8319

FIG 9.3 How does FmetHb S compare to FmetHb A? Tertiary Structure differences between Hb A and Hb S -intensity increase of W3 Trp mode at 1558 cm-1 -stronger H-bond between 15/14 Trp residues and 72 Ser and 67 Thr residues on E-helix -displacement of the A-helix present in both quaternary states

How do Hb S fibers compare with Hb S tetramers?  

FIG 9.4 Quaternary structure of Hb S fibers T-state contacts are stronger in fibers -all Trp modes are more intense -Tyr modes exhibit larger shifts

Our subsequent UVRR studies with Hb S fibers detected many different changes in the protein relative to the individual tetramer molecules (link to pdf of published paper 350 kb).  In terms of the H-bonds that stabilize the T-state, our studies showed that these bonds are stronger in the fibers than they are in the individual tetramers (FIG 9.4).  It has been known for a long time that it is harder to get the fibers to take up oxygen once formed; thus, our finding provides a physical basis for this observation.

The UVRR studies also showed an increase in Phe signal intensity (FIG 9.5).  This increase is consistent with an increase in hydrophobicity of the Phe local environment (FIG 9.6).  

FIG 9.7 UV Resonance Raman Spectrometer

We have assigned this signal to the b85 Phe residue that is in the hydrophobic pocket, which interacts with the b6 mutated residue of a different tetramer (FIG 9.7).  This is one of only two Phe residues in the protein that experiences a different environment as a consequence of fiber formation.

FIG 9.8   Probing secondary structure conformation (F-R) and (F-T) difference spectra  - Amide I and III frequencies indicate increase in random or unordered structure  - Intensity increase of Amide II and C-H indicates loss in helical structure

Other structural changes observed:

·        Decrease in a-helical content (FIG 9.8) and an increase in random coil structure.  The protein could be unfolding to make a better contact in the donor-acceptor interaction.

·        b₂5 Pro residue next to the mutated b₂6 Val residue experiences a weakening in H-bond interaction in the fibers.  This weakening is consistent with a reduction in a-helical structure of the N-terminus and general plasticity of the donor-acceptor interaction.

·        Trp residues are heterogeneous in terms of their local environment.  b37 Trp residues at the subunit interface forms stronger H-bonds (FIG 9.9).  b15 Trp residue is in a very hydrophobic environment, consistent with its proximity to the important ¹b₁-²b₂ interaction (FIG 9.10).

	FIG 9.9 Tyr and Trp residues in Hb Intersubunit T-state H-bonds:   b37 Trp … a94 Asp a42Tyr ... b99 Asp •
FIG 9.10 The 1ß1 - 2ß2 interaction 2b26 Val interacts with 1b185 Phe and 1b188 Leu in the donor-acceptor interaction 2b subunit = donor– 1b subunit = acceptor Monitor b85 Phe by UVRR spectroscopy

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(Hb research in the Mukerji Lab)