Hemoglobin Cooperativity Structural Aspects Of The T R Transition Biology Essay

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The objective of this lab exercise is to study the structural transition that takes place in hemoglobin upon binding of oxygen molecules (the transition from deoxy T state to the oxy R state). The exercise is intended to give experience in some of the techniques used for detailed structural analysis in complex protein systems (~4500 atoms). Specifically you will be using the methods of (least-squares) superposition of co-ordinates and graphical structural examination. The intelligent use of atom selection, colouring and orientation in the graphics session will enhance the extraction of relevant information.

Hemoglobin is known to undergo a structural transition, from the deoxy T state to the oxy R state upon binding of oxygen molecules. This R-T transition stands at the core of hemoglobin co-operative activity. In this exercise we examine the structure of adult human hemoglobin (HbA) in the deoxy (T) and oxy (R) states. By comparison of the structures we observe the changes that occur on oxygenation of hemoglobin, both in tertiary structure (within a subunit) and in quaternary structure (between subunits). This will allow us to visualise components of the proposed structural mechanism of hemoglobin co-operativity.

The unveiling of Hemoglobin's co-operative mechanism has come from the study of a number of different hemoglobins, crystallized under different conditions and with various ligands bound to them. Due to their different ligand saturation levels and quaternary structural arrangement, some of these hemoglobins represent intermediate 'states' between the classic R and classic T structures. Here, for simplicity, we restrict ourselves to two structures: a deoxy R conformation and a fully oxygenated T conformation. These are sufficient to illustrate the main structural phenomena. This laboratory exercise is divided into four sections, in which you will:

a) Become acquainted with the overall arrangement of the molecule (the alpha-1 subunit, the alpha-1/beta-1 dimer and the tetramer).

b) Learn how to superimpose structures in the appropriate reference frame for comparison.

c) Study tertiary structure changes (in the alpha-1 and beta-1 subunits).

d) Study quaternary structure changes (in the alpha-1/beta-2 interface and in the alpha-1/alpha-2 and beta-1/beta-2 interfaces).

Procedure

The X-ray co-ordinates sets that you will use in this lab are:

Deoxy (T): Deoxy HbA at 1.7Å resolution (from the PDB entry pdb2hhb.ent). Reference: Fermi et al. J. Mol. Biol. 175 (1984), 159.

Oxy (R): HbA with oxygen bound at 2.1Å resolution. (from the PDB entry bio1hho.pdb) Reference: Shaanan, J. Mol. Biol. 171 (1983), 31.

Overall Arrangement of the Molecule

The alpha-1 subunit:

Read in (Open PDB) the deoxy structure

Create a display selection to show the backbone (N, Ca, C) atoms of the globin, the heme and the proximal histidine (alp1:f8) of the alpha-1 subunit (protein A).

Choose a suitable color scheme to distinguish globin from heme!

Examine the structure. Look at the arrangement of the helices, the position of the heme and the heme-globin linkage.

Change the display selection to view all of the alpha-1 subunit atoms (too much information?)

Change the display selection to view all residues with atoms within 7<81> of the ND1 atom of the distal histidine (E7) Color by atom

Q1:List the residues that comprise the distal enviroment (and their type). Comment on the nature of the heme environment.

The alpha-1/beta-1 dimer:

Modify the selections to include both the alpha-1 and beta-1 subunit backbone atoms (colour accordingly).

Q2: Which secondary structure elements of the subunits make up the inferface?

The tetramer:

Modify the above selections to examine the complete tetramer.

Use an appropriate color selection to distinguish alpha and beta subunits.

Look at the arrangement of the subunits and the various subunit interfaces

Q3: What are the distances between the iron atoms in the 4 subunits? (get a feeling for the dimensions of this molecular assembly).

B. Superposition of the Structures in the BGH Frame

As the structural differences of R and T involve both tertiary and quaternary structure changes it is essential that a suitable reference frame be selected for their comparison (why?). Baldwin and Chothia showed that the structure in the region of contact at the alpha-1/beta-1 interface is the same in R and T structures. If R and T structures are superposed in this region, they showed that the differences between alpha-1 and beta-1 reflect tertiary structural changes and that differences between alpha-2 and beta-2 reflect both tertiary and quaternary structural changes. The region for superposition includes residues from B, G and H helices and thus this particular frame of reference is known as BGH, and is commonly used.

Load both oxy and deoxy structures.

Create an atom selection for all atoms in both structures, change display to show only backbone.

Make each structure a different color.

Transform the molecules into the GBH frame by superposing the alpha-carbons of the follwing residues: B4-B14, G6-G18 and H5-H17, in both alpha-1 (A) and beta-1 (B) subunits; giving a total of 74 atom matches overall.

Q4: What is the RMS co-ordinate difference in this region?

C. Tertiary Structure Changes

Examining the T/R differences in the individual subunits. Use the transformed oxy co-ordinates (or stay within the comparison application).

The alpha-1 subunit:

Create a display selection for the alpha-1 subunits in both the deoxy and oxy states, to show the backbone atoms of the globin and the heme.

Q5: What is the behaviour of the iron atom? Which regions of backbone differ most?

Change the display to the following: Show the backbone of the globin from residue E1 to residue G1, the Heme and show all atoms for residues F8, FG3, FG5, C7, CD1, CD4, E7 and E11 (the ligand in the oxy structure is included in the heme residue ). The displayed backbone serves as a reference frame for the specific residues we like to focus on.

Notice the movement of the proximal histidine (F8) associated with the heme

Notice the movement in the sidechains of Leu FG3 and Val FG5.

Notice how the structure of the distal residues is largely the same in the two structures.

Q6: The heme, His F8, the H-helix and the FG corner have been described as the allosteric core of hemoglobin (Gelin, Lee and Karplus, J. Mol. Biol., 171, 489 (1983)). Why ?

The beta-1 subunit:

arrange for the selection of the residues specified above showing the allosteric core and distal residues in the beta-1 subunit.

Q7: What are the major differences you observe from the alpha-1 subunit ?

D. Quaternary Structure Changes

Examining the T/R differences between the subunits.

Again use the transformed oxy co-ordinates.

The alpha-1/beta-2 interface:

Create a display selection to show atoms of alpha-1 and beta-2 subunits.

Color the FG corners, C helices and HC termini of each subunit the same, but distinct from one another. Make everything else the same colour.

See how the FG corner of alpha-1 and the C helix of beta-2 remain similar in packing arrangement and how the C helix of alpha-1 and the FG corner of beta-2 differ. These regions have been described as the joint and switch of the quaternary structure changes.

Q8: To what specific structural feature does the term switch refer? Can you see it? How do the quaternary shifts effect specific interactions at the interface, how is this important in the co-operative mechanism?

The alpha-1/alpha-2 and beta-1/beta-2 interfaces

Display the complete tetramer.

Q9: What is the major effect upon going from T to R on the central cavity of the tetramer ?

TIP: Flash between the two structures.

Writeup

Answer all the questions presented throughout the lab and in addition answer:

Q10: What is an allosteric effector? Give examples and describe their principal binding sites.

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