Hemoglobin & Sickle Cell Anemia Exercise

Name______

StarBiochem

Hemoglobin & Sickle Cell Anemia Exercise

Learning Objectives

In this exercise, you will use StarBiochem, a protein 3D viewer, to explore:

• the structure of the hemoglobin protein
• the structure of the abnormal form of hemoglobin (HbS) that results in sickle cell anemia, a genetically inherited blood disorder

• the specific amino acid substitution in HbS that causes sickle cell anemia

Background

Hemoglobin is the protein in red blood cells responsible for carrying oxygen from the lung to the rest of the body and for returning carbon dioxide from the rest of the body to the lung. Hemoglobin is comprised of four protein chains. Each of these chains contains a “heme” group. Each heme group can bind to an oxygen (O2) molecule.

Sickle cell anemia is a genetic disorder. A single amino acid substitution in a specific protein chain of hemoglobin is responsible for the development of sickle cell anemia. This single amino acid substitutionresults in the adherence of hemoglobin proteins to each other, forming long cables that can distort normal red blood cells into sickle shaped cells. Unlike normal red blood cells which are flexible and can fit through small capillaries, sickled red blood cells are unflexible and have the tendency to clog blood vessels. This can resultin organ damage and pain in individuals with sickle cell anemia.

The prevalance of this disorder in United States is approximately 1 in 5000 individuals, and it mostly affects African Americans, South Asians and Hispanics.

Getting started with StarBiochem

• To being using StarBiochem, please navigate to: .
• Click on the Start button to launch the application.
• Click Trust when a prompt appears asking if you trust the certificate.

• Under Samples, select “1A3N”(the four letter ID for normal human hemoglobin).

Take a moment to look at the structure from various angles by rotating andzooming on the structure.

• Instructions for changing the view of structure can be found in the top menu, under View -> Structure viewing instruction.

Page 2 contains a series of terms and useful information that you will refer to during this exercise. Briefly, review the terms on this page before proceeding.

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Ver. 6 - M. Rokop, D. Sinha and L. Alemán

Reference

Chemical Structures of the Amino Acids

The 20 amino acids share a common backbone and are distinguished by different side chains, also called ‘R’ groups, highlighted by the various colors below.

PROTEIN STRUCTURE BASICS

All proteins have the following three levels of protein structure:

Primary structure

Describes the order of the amino acids in the protein chain but does not describe its shape.

Secondary structure

Describes shapes that form from local folding of regions within the amino acid chain. These smaller structures can be divided into two main types: helices and sheets. Coils are made of amino acids that do not form regular secondary structures (helices and sheets) but play important roles in protein folding.

Tertiary structure

Describes the entire folded shape of a protein chain.

In addition, some proteins interact with themselves or with other proteins to form larger protein structures. These proteins have an additional level of protein structure:

Quaternary structure

Describes how multiple protein chains interact and fold to form a larger protein complex.

Exercise

We will first explore the structure of normal hemoglobin and then explore the structure of sickle hemoglobin to reveal how a single amino acid in this protein leads to the development of sickle cell anemia.

Protein Structure Questions

1How many amino acids does normal hemoglobin (1A3N) have?

Note: hemoglobin contains four protein chains.

• Click on Primary.

• The sequence of amino acids, including their identity and position withineachprotein chain, can be found within the amino acid sequence box.

Answer

2Briefly look at the primary sequence of each protein chain.Are the protein chains within normal hemoglobin (1A3N) likely to be identical or different?AnswerYes/No and provide a brief explanation for your answer.

Answer

3In addition to amino acids, each protein chain in hemoglobin also contains a chemical groupcalled heme, which binds to oxygen.Which chemical elements comprise heme groups? How many atoms do you find for eachchemical element in eachheme group?

• Within the Primary tab, make sure all the amino acids in hemoglobin have been selected (there should all be highlighted in blue). Move the Atoms Sizeslider completely to the left and the Bonds Translucencyslider to the right (85%) to minimize the appearance of all the amino acids in the hemoglobin protein.
• If desired, you can increase the size of the atoms found in the heme groups by clicking on the HeteroAtoms tab and by moving the Atoms Size slider to the right.

• Gray = Carbon, Blue = Nitrogen, Red = Oxygen, Orange = Iron

Answer

4Tertiary and quaternary structure are formed by the bending and folding of polypeptide chains. These levels of protein structure are stabilized by various covalent and non-covalent interactions between the side-chains of different amino acids.We will now take a deeper look at two amino acids involved in the tertiary structure of hemoglobin: amino acids 85 and88 in the 2ndprotein chain (protein chain B).Based on the nature of their side-chains, what is the strongest bond type that amino acids 85 and 88 in the 2nd protein chain would form with each other’s side chains? Your choices are ‘an ionic bond’, ‘a hydrogen bond’, ‘a hydrophobic interaction’, ‘a vader Walls interaction’ or ‘a covalent bond’? Explain your choice.

• Optional: These set of steps are not necessary for answering this question but will illustrate how you can use StarBiochem to visualize the interaction between these two amino acids.

• Within the Protein tab, click on the Tertiarytab.
• Within the amino acid sequence window, select amino acids 85 and 88 only: click on amino acid 85, hold down Control andApple (Apple)/Control and Command (PC)and then click on amino acid 88.
• Explore the category these two amino acids belong to by clicking on the different amino acid types choices within the Tertiarytab.

Answer

Structure -> Function -> Disease Questions

5We will now take a look at the structure of sickle hemoglobin (2HBS), and compare its structure to that of normal (wild type) hemoglobin (1A3N), to understand how a single amino acid change in hemoglobin leads to sickle cell anemia.

• In the top menu, under View, click on Reset structure.
• Click on View and choose Reset Molecule.

• Under Samples, select “2HBS”(the four letter ID for sickle human hemoglobin). A new tab will appear showing the 2HBS structure right next to the 1A3N structure tab.

a)How does the overall structure of normal hemoglobin (1A3N) differ from that of sickle hemoglobin (2HBS)?Explain in your own words.

Answer

b)Circle the correct statement(s) from the options below.

The single amino acid substitution in sickle hemoglobin:

Answer

influences the overall structure of anindividual hemoglobin protein

does not influence the overall structure of an individual hemoglobin protein

6A single amino acid substitution at position 6within a protein chain(s) of hemoglobin to the amino acid valine results in sickle cell anemia.

a)Identify the protein chain(s) in sickle human hemoglobin (2HBS) where you observe the presence of thisspecific amino acid substitution.

Answer

b)Name the amino acid present in normal human hemoglobin (1A3N) that is substituted by valineat position 6in sickle human hemoglobin (2HBS).

Answer

7In the sickle human hemoglobin structure (2HBS)valine 6 in one protein chain of one of the two hemoglobin proteins interacts with phenylalanine85 and leucine88 located in the protein chain of another sickle human hemoglobin protein.

a) Identify the protein chains (i.e. A, B, C, D) that contain these three amino acids in a configuration that allows them to interact with each other.

• Under Structure click onPrimary.
• Select more than one amino acid residue by by individually clicking on them and simultaneously pressing Control and Apple key(Mac)/right-click (PC).

• The amino acids you select get highlighted in the structure (white). For a better view you can go to View Controls and move the Unselectedtransparency slider to “0”.

Answer

b)What is the most likely interaction between valine 6 and phenylalanine 85 and leucine 88? Please explain.

Answer

c)In question 8 (b) of this exercise you have identified the amino acid located at position #6 in normal hemoglobin, Hb (1A3N). This amino acid, unlike valine 6 in sickle hemoglobin (2HBS), does not interact with phenylalanine 85 and leucine 88. Propose an explanation for this observation.

Answer

d)Based on what you have learned from this exercise, explain whyan amino acid substitution to valineat position 6 results in sickle cell anemia.

Answer

Keywords

Sickle cell anemia, essential amino acids, oxyhemoglobin or saturated hemoglobin, deoxyhemoglobin or desaturated hemoglobin, and autosomal recessive genetic disorder.

Thought Questions

1Sickle cell patients are very often asked to avoid dehydration by significantly increasing their fluid intake. Explain how this recommendation may help these patients.

2Abnormalities in the hemoglobin protein account for a variety of genetically inherited disorders such as sickle cell anemia and thalassemia. The genetic mutations responsible for these diseases are much more common in certain regions of the world, i.e. Africa, Eastern Europe and South East Asia. Propose how nature could have selected for the mutant copy of the hemoglobin gene in certain regions of the world.

3World class tennis player, Peter Sampras, and football star ZinedineZidane are thalassemia carriers. These players perform much better in short versus long lasting matches. Based on what you have learned about hemoglobin from this exercise, explain why this may be so.

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Ver. 6 - M. Rokop, D. Sinha and L. Alemán