Hemoglobin: Soup to Nuts

"A discovery is like falling in love and reaching the top of a mountain after a hard climb all in one, an ecstasy induced not by drugs but the revelation of a face of nature that no one has seen

before" -Max Perutz

The Issue• How to use one data set to answer multiple

questions in multiple classes– Introduce the data set and a simple problem in

General Biology (Introductory Cell Biology and Genetics)

– Expand the depth and sophistication of questions (but use the same data set!) for more advanced courses (Genetics, Microbiology, Molecular Biology, Biochemistry, Evolution….)

The Answer

• Hemoglobin!• DNA and protein sequences are available for the

various globin proteins (alpha, beta, embryonic, fetal, adult, disease-causing mutants, plant leghemoglobin)

• Crystal structures are available for various globin proteins

• Initial time spent setting up data set will pay off in multiple classes AND prevent the Las Vegas syndrome many students exhibit

The Las Vegas Syndrome

• “What happens in Vegas stays in Vegas.”

• Students seem reluctant to bring knowledge from one class to another class

• Using a single molecule for various types of analysis in several classes may help cure the dreaded L.V. Syndrome

Hemoglobin for Introductory Cell Biology and Genetics

• Hemoglobins are the oxygen-carrying proteins in red blood cells. They are tetrameric (4-chain) proteins containing two copies each of two different polypeptide chains, which is called the structure. In addition each polypeptide chain contains a heme group, which is a ring-shaped organic molecule in which iron is embedded and to which oxygen is bound.

• In humans there are in fact two groups of closely related genes encoding the alpha chain family on Chromosome 16 and the beta chain family on Chromosome 11. Different alpha and beta chain family members are expressed at different times during normal human development. There are embryonic alpha and beta forms that are expressed only during the first 6 to 8 weeks of gestation, after which there is a shift to expression of the adult form of the alpha chain and fetal forms of the beta chain. At about the time of birth adult forms of the beta chains also appear, and these "adult" forms of both chains are normally expressed throughout the remainder of an individual's life. The embryonic and fetal forms of hemoglobin bind oxygen more strongly than the adult form, ensuring that the fetus obtains adequate oxygen in the womb.

Globin subunit

Heme group

Oxygen (red)

Introduction to workbench

• Use ClustalW to align an alpha sequence and a beta sequence

• Explain rooted and unrooted tree function

The exercise

• Download file of normal embryonic, fetal and adult hemoglobin amino acid sequences

• Perform an alignment using ClustalW

• Determine which sequences are alpha-like and which sequences are beta-like

>Human_alpha chainMVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

>Human beta chainMVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

>Human epsilon chainMVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFGKEFTPEVQAAWQKLVSAVAIALAHKYH

>Human epsilon globinMVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFGKEFTPEVQAAWQKLVSAVAIALAHKYH

>Human zeta globinMSLTKTERTIIVSMWAKISTQADTIGTETLERLFLSHPQTKTYFPHFDLHPGSAQLRAHGSKVVAAVGDAVKSIDDIGGALSKLSELHAYILRVDPVNFKLLSHCLLVTLAARFPADFTAEAHAAWDKFLSVVSSVLTEKYR

>Human delta globinMVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANALAHKYH

>HUMAN gamma-AMGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRY

>Human gamma globinMGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH

Alignment

Trees

Debriefing

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

http://www.people.virginia.edu/~rjh9u/globinevolve.html

Future topics

• Genetics, Evolution– Abnormal hemoglobins (thalassemias, sickle cell), selective

pressures

• Microbiology– Relationship of hemoglobin to leghemoglobin, differences

in structure and function

• Animal Physiology– Comparison of hemoglobins across taxa, oxygen binding

coefficients as a function of sequence

• Biochemistry– Oxygen loading, cooperativity