ROTEINS
`
`STRUCTURE AND FUNCTION
`
`David Whitford
`
`John Wiley & Sons, Ltd
`
`1 of 209
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`Fresenius Kabi
`Exhibit 1009
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 3 of 43
`
`Copyright © 2005
`
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`2 of 209
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 4 of 43
`
`Contents
`
`Preface
`
`1 An Introduction to protein structure and function
`A brief and very selective historical perspective
`The biological diversity of proteins
`Proteins and the sequencing of the human and other genomes
`Why study proteins?
`
`2 Amino acids: the building blocks of proteins
`The 20 amino acids found in proteins
`The acid-base properties of amino acids
`Stereochemical representations of amino acids
`Peptide bonds
`The chemical and physical properties of amino acids
`Detection, identification and quantification of amino acids and proteins
`Stereoisomerism
`Non-standard amino acids
`Summary
`Problems
`
`3 The three-dimensional structure of proteins
`Primary structure or sequence
`Secondary structure
`Tertiary structure
`Quaternary structure
`The globin family and the role of quaternary structure in modulating activity
`Immunoglobulins
`Cyclic proteins
`Summary
`Problems
`
`4 The structure and function of fibrous proteins
`The amino acid composition and organization of fibrous proteins
`Keratins
`Fibroin
`Collagen
`Summary
`Problems
`
`xi
`
`1
`1
`5
`9
`9
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`13
`13
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 5 of 43
`
`5 The structure and function of membrane proteins
`The molecular organization of membranes
`Membrane protein topology and function seen through organization of the
`erythrocyte membrane
`Bacteriorhodopsin and the discovery of seven transmembrane helices
`The structure of the bacterial reaction centre
`Oxygenic photosynthesis
`Photosystem I
`Membrane proteins based on transmembrane ~ barrels
`Respiratory complexes
`Complex III, the ubiquinol-cytochrome c oxidoreductase
`Complex IV or cytochrome oxidase
`The structure of ATP synthetase
`ATPase family
`Summary
`Problems
`
`6 The diversity of proteins
`Prebiotic synthesis and the origins of proteins
`Evolutionary divergence of organisms and its relationship to protein
`structure and function
`Protein sequence analysis
`Protein databases
`Gene fusion and duplication
`Secondary structure prediction
`Genomics and proteomics
`Summary
`Problems
`
`7 Enzyme kinetics, structure, function, and catalysis
`Enzyme nomenclature
`Enzyme co-factors
`Chemical kinetics
`The transition state and the action of enzymes
`The kinetics of enzyme action
`Catalytic mechanisms
`Enzyme structure
`Lysozyme
`The serine proteases
`Triose phosphate isomerase
`Tyrosyl tRNA synthetase
`EcoRI restriction endonuclease
`Enzyme inhibition and regulation
`Irreversible inhibition of enzyme activity
`Allosteric regulation
`Covalent modification
`Isoenzymes or isozymes
`Summary
`Problems
`
`105
`105
`
`110
`114
`123
`126
`126
`128
`132
`132
`138
`144
`152
`156
`159
`
`161
`161
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`163
`165
`180
`181
`181
`183
`187
`187
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`189
`191
`192
`192
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`197
`202
`209
`209
`212
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 6 of 43
`
`8 Protein synthesis, processing and turnover
`Cell cycle
`The structure of Cdk and its role in the cell cycle
`Cdk-cyclin complex regulation
`DNA replication
`Transcription
`Eukaryotic transcription factors: variation on a 'basic' theme
`The spliceosome and its role in transcription
`Translation
`Transfer RNA (tRNA)
`The composition of prokaryotic and eukaryotic ribosomes
`A structural basis for protein synthesis
`An outline of protein synthesis
`Antibiotics provide insight into protein synthesis
`Affinity labelling and RNA 'footprinting'
`Structural studies of the ribosome
`Post-translational modification of proteins
`Protein sorting or targeting
`The nuclear pore assembly
`Protein turn over
`Apoptosis
`Summary
`Problems
`
`9 Protein expression, purification and characterization
`The isolation and characterization of proteins
`Recombinant DNA technology and protein expression
`Purification of proteins
`Centrifugation
`Solubility and 'salting out' and 'salting in'
`Chromatography
`Dialysis and ultrafiltration
`Polyacrylamide gel electrophoresis
`Mass spectrometry
`How to purify a protein?
`Summary
`Problems
`
`10 Physical methods of determining the three-dimensional structure of
`proteins
`Introduction
`The use of electromagnetic radiation
`X-ray crystallography
`Nuclear magnetic resonance spectroscopy
`Cryoelectron microscopy
`Neutron diffraction
`Optical spectroscopic techniques
`Vibrational spectroscopy
`Raman spectroscopy
`
`247
`247
`250
`252
`253
`254
`261
`265
`266
`267
`269
`272
`273
`278
`279
`279
`287
`293
`302
`303
`310
`310
`312
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`313
`313
`313
`318
`320
`323
`326
`333
`333
`340
`342
`344
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 7 of 43
`
`ESR and END0R
`Summary
`Problems
`
`11 Protein folding in vivo and in vitro
`Introduction
`Factors determining the protein fold
`Factors governing protein stability
`Folding problem and Levinthal's paradox
`Models of protein folding
`Amide exchange and measurement of protein folding
`Kinetic barriers to refolding
`In vivo protein folding
`Membrane protein folding
`Protein misfolding and the disease state
`Summary
`Problems
`
`12 Protein structure and a molecular approach to medicine
`Introduction
`Sickle cell anaemia
`Viruses and their impact on health as seen through structure and function
`HIV and AIDS
`The influenza virus
`p53 and its role in cancer
`Emphysema and ct1-antitrypsin
`Summary
`Problems
`
`Epilogue
`
`Glossary
`
`Appendices
`
`Bibliography
`
`References
`
`Index
`
`390
`392
`393
`
`395
`395
`395
`403
`403
`408
`411
`412
`415
`422
`426
`435
`437
`
`439
`439
`441
`442
`443
`457
`470
`475
`478
`479
`
`481
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`483
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 8 of 43
`
`An Introduction to protein structure
`and function
`
`Biochemistry has exploded as a major scientific
`endeavour over the last one hundred years to rival pre(cid:173)
`viously established disciplines such as chemistry and
`physics. This occurred with the recognition that living
`systems are based on the familiar elements of organic
`chemistry (carbon, oxygen, nitrogen and hydrogen)
`together with the occasional involvement of inorganic
`chemistry and elements such as iron, copper, sodium,
`potassium and magnesium. More importantly the laws
`of physics including those concerning thermodynam(cid:173)
`ics, electricity and quantum physics are applicable to
`biochemical systems and no 'vital' force distinguishes
`living from non-living systems. As a result the laws
`of chemistry and physics are successfully applied to
`biochemistry and ideas from physics and chemistry
`have found widespread application, frequently revolu(cid:173)
`tionizing our understanding of complex systems such
`as cells.
`This book focuses on one major component of all
`living systems - the proteins. Proteins are found in
`all living systems ranging from bacteria and viruses
`through the unicellular and simple eukaryotes to
`vertebrates and higher mammals such as humans.
`Proteins make up over 50 percent of the dry weight
`of cells and are present in greater amounts than
`any other biomolecule. Proteins are unique amongst
`the macromolecules in underpinning every reaction
`
`Proteins: Structure and Function by David Whitford
`© 2005 John Wiley & Sons, Ltd
`
`occurring in biological systems. It goes without saying
`that one should not ignore the other components of
`living systems since they have indispensable roles, but
`in this text we will consider only proteins.
`
`A brief and very selective historical
`perspective
`
`With the vast accumulation of knowledge about pro(cid:173)
`teins over the last 50 years it is perhaps surprising to
`discover that the term protein was introduced nearly
`170 years ago. One early description was by Gerhardus
`Johannes Mulder in 1839 where his studies on the com(cid:173)
`position of animal substances, chiefly fibrin, albumin
`and gelatin, showed the presence of carbon, hydro(cid:173)
`gen, oxygen and nitrogen. In addition he recognized
`that sulfur and phosphorus were present sometimes in
`'animal substances' that contained large numbers of
`atoms. In other words, he established that these 'sub(cid:173)
`stances' were macromolecules. Mulder communicated
`his results to Jons Jakob Berzelius and it is suggested
`the term protein arose from this interaction where the
`origin of the word protein has been variously ascribed
`to derivation from the Latin word primarius or from
`the Greek god Proteus. The definition of proteins was
`timely since in 1828 Friedrich Wohler had shown that
`
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`2
`
`AN INTRODUCTION TO PROTEIN STRUCTURE ANO FUNCTION
`
`Figure 1.1 The decomposition of ammonium cyanate
`yields urea
`
`heating ammonium cyanate resulted in isomerism and
`the formation of urea (Figure 1.1). Organic compounds
`characteristic of living systems, such as urea, could
`be derived from simple inorganic chemicals. For many
`historians this marks the beginning of biochemistry and
`it is appropriate that the discovery of proteins occurred
`at the same period.
`The development of biochemistry and the study of
`proteins was assisted by analysis of their composition
`and structure by Heinrich Hlasiwetz and Josef Haber(cid:173)
`mann around 1873 and the recognition that proteins
`were made up of smaller units called amino acids.
`They established that hydrolysis of casein with strong
`acids or alkali yielded glutamic acid, aspartic acid,
`leucine, tyrosine and ammonia whilst the hydrolysis
`of other proteins yielded a different group of products.
`Importantly their work suggested that the properties of
`proteins depended uniquely on the constituent parts - a
`theme that is equally relevant today in modern bio(cid:173)
`chemical study.
`Another landmark in the study of proteins occurred
`in 1902 with Franz Hofmeister establishing the con(cid:173)
`stituent atoms of the peptide bond with the polypep(cid:173)
`tide backbone derived from the condensation of free
`amino acids. Five years earlier Eduard Buchner rev(cid:173)
`olutionized views of protein function by demonstrat(cid:173)
`ing that yeast cell extracts catalysed fermentation of
`sugar into ethanol and carbon dioxide. Previously it
`was believed that only living systems performed this
`catalytic function. Emil Fischer further studied biolog(cid:173)
`ical catalysis and proposed that components of yeast,
`which he called enzymes, combined with sugar to pro(cid:173)
`duce an intermediate compound. With the realization
`that cells were full of enzymes 100 years of research
`has developed and refined these discoveries. Further
`landmarks in the study of proteins could include Sum(cid:173)
`ner's crystallization of the first enzyme (urease) in
`1926 and Pauling's description of the geometry of the
`
`peptide bond; however, extensive discussion of these
`advances and many other important discoveries in pro(cid:173)
`tein biochemistry are best left to history of science
`textbooks.
`A brief look at the award of the Nobel Prizes
`for Chemistry, Physiology and Medicine since 1900
`highlighted in Table 1.1 reveals the involvement of
`many diverse areas of science in protein biochemistry.
`At first glance it is not obvious why William and
`Lawrence Bragg's discovery of the diffraction of
`X-rays by sodium chloride crystals is relevant, but
`diffraction by protein crystals is the main route towards
`biological structure determination. Their discovery was
`the first step in the development of this technique.
`Discoveries in chemistry and physics have been
`implemented rapidly in the study of proteins. By 1958
`Max Perutz and John Kendrew had determined the first
`protein structure and this was soon followed by the
`larger, multiple subunit, structure of haemoglobin and
`the first enzyme, lysozyme. This remarkable advance
`in knowledge extended from initial understanding of
`the atomic composition of proteins around 1900 to
`the determination of the three-dimensional structure of
`proteins in the 1960s and represents a major chapter
`of modern biochemistry. However, advances have
`continued with new areas of molecular biology proving
`equally important to understanding protein structure
`and function.
`Life may be defined as the ordered interaction
`of proteins and all forms of life from viruses to
`complex, specialized, mammalian cells are based on
`proteins made up of the same building blocks or
`amino acids. Proteins found in simple unicellular
`organisms such as bacteria are identical in structure
`and function to those found in human cells illustrating
`the evolutionary lineage from simple to complex
`organisms.
`Molecular biology starts with the dramatic eluci(cid:173)
`dation of the structure of the DNA double helix by
`James Watson, Francis Crick, Rosalind Franklin and
`Maurice Wilkins in 1953. Today, details of DNA repli(cid:173)
`cation, transcription into RNA and the synthesis of pro(cid:173)
`teins (translation) are extensive. This has established
`an enormous body of knowledge representing a whole
`new subject area. All cells encode the information con(cid:173)
`tent of proteins within genes, or more accurately the
`order of bases along the DNA strand, yet it is the
`
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 10 of 43
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`A BRIEF AND VERY SELECTIVE HISTORlCAl PERSPECTIVE
`
`3
`
`fable 1.1 Selected landmarks in the study of protein structure and function from 1900-2002 as seen by the award
`of the Nobel Prize for Chemistry, Physiology or Medicine
`
`Date
`
`1901
`
`1907
`1914
`1915
`
`1923
`1930
`1946
`
`1948
`
`1952
`
`1952
`
`1954
`
`1958
`1959
`
`1962
`
`1962
`
`1964
`
`1965
`
`1968
`
`1969
`
`Discoverer + Discovery
`
`Wilhelm Conrad Rontgen 'in recognition of the ... discovery of the remarkable rays subsequently
`named after him'
`Eduard Buchner 'cell-free fermentation'
`Max von Laue 'for his discovery of the diffraction of X-rays by crystals'
`William Henry Bragg and William Lawrence Bragg 'for their services in the analysis of crystal
`structure by ... X-rays'
`Frederick Grant Banting and John James Richard Macleod 'for the discovery of insulin'
`Karl Landsteiner 'for his discovery of human blood groups'
`James Batcheller Sumner 'for his discovery that enzymes can be crystallized'.
`John Howard Northrop and Wendell Meredith Stanley 'for their preparation of enzymes and virus
`proteins in a pure form'
`Arne Wilhelm Kaurin Tiselius 'for his research on electrophoresis and adsorption analysis, especially
`for his discoveries concerning the complex nature of the serum proteins'
`Archer John Porter Martin and Richard Laurence Millington Synge 'for their invention of partition
`chromatography'
`Felix Bloch and Edward Mills Purcell 'for their development of new methods for nuclear magnetic
`precision measurements and discoveries in connection therewith'
`Linus Carl Pauling 'for his research into the nature of the chemical bond and ... to the elucidation of
`... complex substances'
`Frederick Sanger 'for his work on the structure of proteins, especially that of insulin'
`Severo Ochoa and Arthur Kornberg 'for their discovery of the mechanisms in the biological synthesis
`of ribonucleic acid and deoxyribonucleic acid'
`Max Ferdinand Perutz and John Cowdery Kendrew 'for their studies of the structures of globular
`proteins'
`Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins 'for their
`discoveries concerning the molecular structure of nucleic acids and its significance for information
`transfer in living material'
`Dorothy Crowfoot Hodgkin 'for her determinations by X-ray techniques of the structures of important
`biochemical substances'
`Fran~ois Jacob, Andre Lwoff and Jacques Monod 'for discoveries concerning genetic control of
`enzyme and virus synthesis'
`Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg 'for ... the genetic code and its
`function in protein synthesis'
`Max Delbriick, Alfred D. Hershey and Salvador E. Luria 'for their discoveries concerning the
`replication mechanism and the genetic structure of viruses'
`
`(continued overleaf)
`
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 11 of 43
`
`4
`
`AN INTROOUCTION TO PROTEltl STRUCTURE ANO f\JNCTION
`
`Table L1
`
`(continued)
`
`Date
`
`1972
`
`1972
`
`1975
`
`1975
`
`1978
`
`1980
`
`1982
`
`1984
`
`1984
`
`1988
`
`1989
`
`1991
`
`1992
`
`1993
`
`1994
`
`Discoverer + Discovery
`------ ·-----------~----~---~-- - - -
`---•--·-·-------·-----------------·----
`Christian B. Anfinsen 'for his work on ribonuclease, especially concerning the connection between
`the amino acid sequence and the biologically active conformation' Stanford Moore and William H.
`Stein 'for their contribution to the understanding of the connection between chemical structure and
`catalytic activity of ... ribonuclease molecule'
`Gerald M. Edelman and Rodney R. Porter 'for their discoveries concerning the chemical structure of
`antibodies'
`John Warcup Cornforth 'for his work on the stereochemistry of enzyme-catalyzed reactions'. Vladimir
`Prelog 'for his research into the stereochemistry of organic molecules and reactions'
`David Baltimore, Renato Dulbecco and Howard Martin Temin 'for their discoveries concerning the
`interaction between tumour viruses and the genetic material of the cell'
`Werner Arber, Daniel Nathans and Hamilton 0. Smith 'for the discovery of restriction enzymes and
`their application to problems of molecular genetics'
`Paul Berg 'for his fundamental studies of the biochemistry of nucleic acids, with particular regard to
`recombinant-DNA' Walter Gilbert and Frederick Sanger 'for their contributions concerning the
`determination of base sequences in nucleic acids'
`Aaron Klug 'development of crystallographic electron microscopy and structural elucidation of
`nucleic acid-protein complexes'
`Robert Bruce Merrifield 'for his development of methodology for chemical synthesis on a solid
`matrix'
`Niels K. Jerne, Georges J.F. Kohler and Cesar Milstein 'for theories concerning the specificity in
`development and control of the immune system and the discovery of the principle for production of
`monoclonal antibodies'
`Johann Deisenhofer, Robert Huber and Hartmut Michel 'for the determination of the structure of a
`photosynthetic reaction centre'
`J. Michael Bishop and Harold E. Varmus 'for their discovery of the cellular origin of retroviral
`oncogenes'
`Richard R. Ernst 'for ... the methodology of high resolution nuclear magnetic resonance
`spectroscopy'
`Edmond H. Fischer and Edwin G. Krebs 'for their discoveries concerning reversible protein
`phosphorylation as a biological regulatory mechanism'
`Kary B. Mullis 'for his invention of the polymerase chain reaction (PCR) method' and Michael Smith
`'for his fundamental contributions to the establishment of oligonucleotide-based, site-directed
`mutagenesis'
`Alfred G. Gilman and Martin Rodbell 'for their discovery of G-proteins and the role of these proteins
`in signal transduction'
`
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`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 12 of 43
`
`.
`.
`THE BIOLOGICAL DIVERSITY OF PROTEINS
`
`S
`
`Table 1.1
`
`(continued)
`
`Date
`
`1997
`
`1997
`1999
`
`2000
`2001
`2002
`
`Discoverer + Discovery
`
`.......... , __ .. ___ ·---------·--·--------
`Paul D. Boyer and John E. Walker 'for their elucidation of the enzymatic mechanism underlying the
`synthesis of adenosine triphosphate (ATP)'. Jens C. Skou 'for the first discovery of an
`ion-transporting enzyme, Na+, K+ -ATPase'
`Stanley B. Prusiner 'for his discovery of prions - a new biological principle of infection'
`GUnter Blobel 'for the discovery that proteins have intrinsic signals that govern their transport and
`localization in the cell'
`Arvid Carlsson, Paul Greengard and Eric R Kandel 'signal transduction in the nervous system'
`Paul Nurse, Tim Hunt and Leland Hartwill 'for discoveries of key regulators of the cell cycle'
`Kurt Wuthrich, 'for development of NMR spectroscopy as a method of determining biological
`macromolecules structure in solution.' John B. Fenn and Koichi Tanaka 'for their development of
`soft desorption ionization methods for mass spectrometric analyses of biological macromolecules'.
`Sydney Brenner, H. Robert Horvitz and John E. Sulston 'for their discoveries concerning genetic
`regulation of organ development and programmed cell death'
`
`]
`
`"' 0 j
`I "'
`
`conversion of this information or expression into pro(cid:173)
`teins that represents the tangible evidence of a living
`system or life.
`
`DNA--+ RNA--+ protein
`
`Cells divide, synthesize new products, secrete unwanted
`products, generate chemical energy to sustain these pro(cid:173)
`cesses via specific chemical reactions, and in all of
`these examples the common theme is the mediation
`of proteins.
`In 1944 the physicist Erwin Schrodinger posed the
`question 'What is Life?' in an attempt to understand the
`physical properties of a living cell. Schrodinger sug(cid:173)
`gested that living systems obeyed all laws of physics
`and should not be viewed as exceptional but instead
`reflected the statistical nature of these laws. More
`importantly, living systems are amenable to study using
`many of the techniques familiar to chemistry and
`physics. The last 50 years of biochemistry have demon(cid:173)
`strated this hypothesis emphatically with tools devel(cid:173)
`oped by physicists and chemists rapidly employed in
`biological studies. A casual perusal of Table 1.1 shows
`how quickly methodologies progress from discovery to
`application.
`
`The biological diversity of proteins
`
`Proteins have diverse biological functions ranging from
`DNA replication, forming cytoskeletal structures, trans(cid:173)
`porting oxygen around the bodies of multicellular
`organisms to converting one molecule into another.
`The types of functional properties are almost end(cid:173)
`less and are continually being increased as we learn
`more about proteins. Some important biological func(cid:173)
`tions are outlined in Table 1.2 but it is to be expected
`that this rudimentary list of properties will expand
`each year as new proteins are characterized. A for(cid:173)
`mal demarcation of proteins into one class should not
`be pursued too far since proteins can have multiple
`roles or functions; many proteins do not lend them(cid:173)
`selves easily to classification schemes. However, for
`all chemical reactions occurring in cells a protein is
`involved intimately in the biological process. These
`proteins are united through their composition based on
`the same group of 20 amino acids. Although all pro(cid:173)
`teins are composed of the same group of 20 amino
`acids they differ in their composition - some contain
`a surfeit of one amino acid whilst others may lack
`one or two members of the group of 20 entirely.
`It was realized early in the study of proteins that
`
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`Exhibit 1009
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`

`Case 0:15-cv-61631-JIC Document 76-6 Entered on FLSD Docket 12/11/2015 Page 13 of 43
`
`6
`
`.
`
`AN UITRODUCTtON TO PROTEIN STIUJCTIJRE ANO fUNcnoN
`
`Table 1.2 A selective list of some functional roles for proteins within cells
`
`Function
`
`Enzymes or catalytic proteins
`Contractile proteins
`Structural or cytoskeletal proteins
`Transport proteins
`
`Effector proteins
`Defence proteins
`Electron transfer proteins
`
`Receptors
`Repressor proteins
`Chaperones (accessory folding proteins)
`Storage proteins
`
`Examples
`
`Trypsin, DNA polymerases and ligases,
`Actin, myosin, tubulin, dynein,
`Tropocollagen, keratin,
`Haemoglobin, myoglobin, serum albumin, ceruloplasmin,
`transthyretin
`Insulin, epidermal growth factor, thyroid stimulating hormone,
`Ricin, immunoglobulins, venoms and toxins, thrombin,
`Cytochrome oxidase, bacterial photosynthetic reaction centre,
`plastocyanin, ferredoxin
`CD4, acetycholine receptor,
`Jun, Fos, Cro,
`GroEL, DnaK
`Ferritin, gliadin,
`
`variation in size and complexity is common and the
`molecular weight and number of subunits (polypep(cid:173)
`tide chains) show tremendous diversity. There is no
`correlation between size and number of polypeptide
`chains. For example, insulin has a relative molecu(cid:173)
`lar mass of 5700 and contains two polypeptide chains,
`haemoglobin has a mass of approximately 65 000 and
`contains four polypeptide chains, and hexokinase is
`a single polypeptide chain with an overall mass of
`~ 100 000 (see Table 1.3).
`The molecular weight is more properly referred to
`as the relative molecular mass (symbol M,). This is
`defined as the mass of a molecule relative to 1112th
`the mass of the carbon (1 2C) isotope. The mass of
`this isotope is defined as exactly 12 atomic mass
`units. Consequently the term molecular weight or
`relative molecular mass is a dimensionless quantity
`and should not possess any units. Frequently in this
`and many other textbooks the unit Dalton (equivalent
`to l atomic mass unit, i.e. l Dalton = 1 amu) is used
`and proteins are described with molecular weights of
`5.5 kDa (5500 Daltons). More accurately, this is the
`absolute molecular weight representing the mass in
`grams of I mole of protein. For most purposes this
`becomes of little relevance and the term 'molecular
`
`Table l..3 The molecular masses of proteins together
`with the number of subunits. The term 'subunit' is
`synonymous with the number of polypeptide chains
`and is used interchangeably
`
`___ , ____ , ___ , ________________ _
`
`Protein
`
`Subunits
`
`Molecular
`mass
`_._•---~"-•--•~------•---•--~••--A•-----•-*•"--•--••------••
`Insulin
`5700
`2
`Haemoglobin
`64500
`4
`Tropocollagen
`285000
`3
`Subtilisin
`27500
`Ribonuclease
`12600
`Aspartate
`310000
`transcarbamoylase
`Bacteriorhodopsin
`Hexokinase
`
`1
`12
`
`1
`1
`
`26800
`102000
`
`weight' is used freely in protein biochemistry and in
`this book.
`Proteins are joined covalently and non-covalently
`with other biomolecules including lipids, carbohydrates,
`
`12 of 209
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`,
`
`THE 810LOGICAL DIVERSITY OF PROTEINS
`
`7
`
`nucleic acids, phosphate groups, flavins, heme groups
`and metal ions. Components such as hemes or metal
`ions are often called prosthetic groups. Complexes
`formed between lipids and proteins are lipoproteins,
`those with carbohydrates are called glycoproteins,
`whilst complexes with metal ions lead to metallo(cid:173)
`proteins, and so on. The complexes formed between
`metal ions and proteins increases the involvement of
`elements of the periodic table beyond that expected
`of typical organic molecules (namely carbon, hydro(cid:173)
`gen, nitrogen and oxygen). Inspection of the periodic
`table (Figure 1.2) shows that at least 20 elements have
`been implicated directly in the structure and function
`of proteins (Table 1.4). Surprisingly elements such as
`aluminium and silicon that are very abundant in the
`Earth's crust (8.1 and 25.7 percent by weight, respec(cid:173)
`tively) do not occur in high concentration within cells.
`Aluminium is rarely, if ever, found as part of proteins
`
`whilst the role of silicon is confined to biomineralization
`where it is the core component of shells. The involve(cid:173)
`ment of carbon, hydrogen, oxygen, nitrogen, phospho(cid:173)
`rus and sulfur is clear although the role of other ele(cid:173)
`ments, particularly transition metals, has been difficult
`to establish. Where transition metals occur in proteins
`there is frequently only one metal atom per mole of pro(cid:173)
`tein and led in the past to a failure to detect metal. Other
`elements have an inferred involvement from growth
`studies showing that depletion from the diet leads to
`an inhibition of normal cellular function. For metallo(cid:173)
`proteins the absence of the metal can lead to a loss of
`structure and function.
`Metals such as Mo, Co and Fe are often found
`associated with organic co-factors such as pterin,
`flavins, cobalamin and porphyrin (Figure 1.3). These
`organic ligands hold metal centres and are often tightly
`associated to proteins.
`
`Table 1.4 The involvement of trace elements in the structure and function of proteins
`
`Element
`
`Sodium
`Potassium
`Magnesium
`
`Calcium
`Vanadium
`Manganese
`
`Iron
`
`Cobalt
`Nickel
`Copper
`
`Zinc
`Chlorine
`Iodine
`Selenium
`
`Functional role '
`
`Principal intracellular ion, osmotic balance
`Principal intracellular ion, osmotic balance
`Bound to ATP/GTP in nucleotide binding proteins, found as structural component of
`hydrolase and isomerase enzymes
`Activator of calcium binding proteins such as calmodulin
`Bound to enzymes such as chloroperoxidase.
`Bound to pterin co-factor in enzymes such as xanthine oxidase or sulphite oxidase. Also
`found in nitroge