1、Proteins IProteins 1830 Mulder what remained after removal of sugars, fats, salts, etc.1838 Berzelius “Proteins” from the Greek “of 1st rank or importance”Central Dogma: DNA RNA protein (information flow)DNA, RNA information is in the sequence - linearProtein function is in the 3D structureWhat are
2、proteins?Proteins are linear polymers (chains) of amino acids (more than 50 aa)“Protein”One or more polypeptide chains One polypeptide chain - a monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example
3、, is a heterotetramer It has two alpha chains and two beta chainsProteins - Large and SmallInsulin - A chain of 21 residues, B chain of 30 residues -total mol. wt. of 5,733Glutamine synthetase - 12 subunits of 468 residues each - total mol. wt. of 600,000Connectin proteins - alpha - MW 2.8 million!b
4、eta connectin - MW of 2.1 million, with a length of 1000 nm -it can stretch to 3000 nm!Main Protein ClassesFibrous - extended shape, insoluble (e.g. keratin, collagen, elastin)Globular - compact shape, water soluble (e.g. myoglobin, trypsin)Membranous - often multidomain one lipid soluble (e.g. phot
5、oreaction centre, ATP synthase)Biological Functions of ProteinsProteins are the agents of biological functionEnzymes - RibonucleaseSignal transduction Insulin and its receptorControl of Gene expression-Transcription factorsImmunity-AntibodyTransport and Storage - HemoglobinStructural proteins Hair,
6、CollagenContractile proteins - Actin, MyosinExotic proteins - Antifreeze proteins in fishProteins are: Polypeptides + possibly cofactors, coenzymes, prosthetic groups, other modifications Polypeptides are covalently linked -amino acids Cofactors are non-amino acid components e.g. metal ions like Zn2
7、+ in carboxypeptidase Coenzymes are organic cofactors e.g. nucleotides in lactate dehydrogenase Prosthetic groups are tightly attached cofactors e.g. heme in myoglobinHierarchy of protein structurePrimary Structure (1) : Unique sequence of amino acids: sequence is determined by genetic materialSecon
8、dary Structure (2) : regular repeating structures of the polypeptide chain (i.e. -helices, b b-sheets) ;coiling /folding as a result of hydrogen bondingTertiary Structure (3) : 3-D shape due to bonding of R- groupsQuaternary Structure (4) : association of 2 or more polypeptides; Ex HGB ; not all hav
9、e this level Hierarchy of protein structurePrimarySecondaryTertiaryQuaternaryPrimary- sequenceSecondary- e.g -helix or b-sheetTertiary- 3D shapeQuaternary- subunit organization ( one polypeptide chain)Four levels of protein structurePrimary: amino acid sequenceDue to covalent bondThe Sequence of Ami
10、no Acids in a Proteinis a unique characteristic of every proteinis encoded by the nucleotide sequence of DNAis thus a form of genetic informationis read from the amino terminus to the carboxyl terminus1o structure =linear amino acid sequenceFor the Insulin A chain, the 1o structure is:Gly-Ile-Val-Gl
11、u-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn Insulin was the first polypeptide to be sequenced (by Frederick Sanger in 1953). The sequencing of insulin demonstrated for the first time that proteins are composed of specific, defined amino acid sequences.The amino acid sequenc
12、e of a protein is specified by the gene encoding that proteinDNA: ggc att gtg gaa caa tgc tgt acc agcmRNA: ggc auu gug gaa caa ugc ugu acc agcProtein: Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-SerThe Peptide Bondhas partial (40%) double bond characteris about 0.133 nm long - shorter than a typical single bond
13、 but longer than a double bondDue to the double bond character, the six atoms of the peptide bond group are always planar! is usually found in the trans conformation, but X-Pro is an exceptionN partially positive; O partially negativeThe Coplanar Nature of the Peptide BondSix atoms of the peptide gr
14、oup lie in a plane!Restriction by Amide PlaneAtoms in the peptide bond lie in a plane. Resonance stabilization energy of this planar structure is approximately 88 kJ/mol;Rotation can only occur around the two bonds connected to the C atom;Rotation around the Ca and carbonyl bond is called y (psi);Ro
15、tation around the Ca and nitrogen bond is called f (phi). Rotation of Amide PlanesIf (f,y) are known for all residues, the structure for the entire backbone is known.Some (f,y) are more likely than others in a folded proteinPositive (f,y) values correspond to clockwise rotation around bonds when vie
16、wed from the C. Zerois defined when the C=O or N-H bond bisects the R-C-H angle.(f,y)=(0,180), two carbonyl oxygens are too close;(f,y)=(180,0), two amide groups are overlapping;(f,y)=(0,0), carbonyl oxygen overlaps with amide group;Ramachandran PlotThe plot uses f as horizontal Axis y as vertical a
17、xis. The (f, y) angle for each residue can be entered on the plot. For folded proteins, their (f, y) angles cluster in few regions of the plot. The upper left corner are beta-sheet values and middle left are -helices values. Lines signifies the number of amino acids per turn of helix (+ means right-
18、handed, - left-handed)Classes of Secondary StructureAll these are local structures that are stabilized by hydrogen bonds Alpha helix Other helices Beta sheet (composed of beta strands) Tight turns (aka beta turns or beta bends) Beta bulge The Alpha Helix The alpha helix is a helical structure. All a
19、lpha helices in proteins are right-handed; H-bond patterns of the alpha helix: Alpha helix: Carbonyl oxygen of the ith residue forms H-bond with amide proton of the (i+4)th residue. So there are n-4 H-bonds in a helix of n amino acids; 310 helix: carbonyl oxygen of the ith residue forms H-bond with
20、amide proton of the (i+3)th residue. 3 residues (or 10 atoms) per turn; Proline is not found in -helix except at the beginning of an -helix; Helix propensity of an amino acid is a measure of the likelyhood for the amino acid to be in a helix; Glu, Met, Ala, Leu have high propensities; Examples of -h
21、elical proteins include -keratin (structural proteins) and collagen (fibrous protein); Linus Pauling (Nobel Prize in Chemistry, 1954) figured out the structure of -keratin helix. The carboxyl group of residue n is hydrogen bonded to the backbone amide of the amino acid at position n+4.NCCOR1HHNCCOR2
22、HHNCCOR3HHNCCOR4HHNCCOR5HHThe Alpha HelixKnow these numbers Residues per turn: 3.6 Rise per residue: 1.5 Angstroms Rise per turn (pitch): 3.6 x 1.5A = 5.4 Angstroms The backbone loop that is closed by any H-bond in an alpha helix contains 13 atoms phi = -60 degrees, psi = -45 degrees The non-integra
23、l number of residues per turn was a surprise to crystallographersThe Alpha HelixResidues per turn: 3.6Rise per residue: 1.5 Rise per turn: 5.4 (f,y)=(-60,-45)C=O N-H side chainTotal dipole moment Insulin chain A contains two -helices Schematic view of the cross-section of an -helix. Side chains are
24、shown as green circles. -helices are often amphipathicEINGFDLLRSGHydrophobic FaceHydrophilic Face A helical wheel representation of an amphipathic a-helix from alcohol dehydrogenase is shown. In a helical wheel, a cross-sectional view of the a -helix is drawn as a spiral with amino acids occurring e
25、very 100o along the spiral (360o divided by 3.6 amino acids per turn gives 100o per amino acid).The Beta-Pleated SheetComposed of beta strands Also first postulated by Pauling and Corey, 1951 Strands may be parallel or antiparallel Rise per residue: 3.47 Angstroms for antiparallel strands 3.25 Angst
26、roms for parallel strands Each strand of a beta sheet may be pictured as a helix with two residues per turnAmino acids that tend to be found in b b-strandsValIle Phe TyrTrpThrThe Beta Turn (tight turn, or b-bend)Beta turns connect beta strands and reverse the direction of beta strands; Proline and g
27、lycine have high propensity for beta turns;The carbonyl oxygen of the ith residue forms H-bond with the amide proton of the (i+3)th residue; Tight turn promotes formation of antiparallel beta sheets.The Beta BulgeBeta bulge occurs between normal b-strands. Comprised of two residues on one strand and
28、 one on the other;Bulges cause bending of otherwise straight anti-parallel beta strands; NHOR3NOR2NHOR1NOR0HHNHONONHONOHHNCR0R2R1R3CNBeta bulgeAnti-parallel strandsNHOR3NOR2NHONNOR0HHNHONONHONOHHNCR0R2R1R3CNHOR1R-1Loops connect secondary structural elements togetherTertiary structure The three dimen
29、sional arrangement of all atoms in a given protein is referred to as the proteins tertiary structure. Contrast tertiary structure with secondary structure, which refers primarily with the arrangement of amino acids that are adjacent in the primary structure (sequence), or very close in the primary s
30、tructure. Again, non-covalent forces are those that are most important, although disulfide bonds contribute to tertiary structure. Motif domain tertiary (conformation)Protein ConformationThe tertiary structure that a protein assumes to carry out its physiological role inside a cell is known as the n
31、ative state or sometimes the native conformationNeed to distinguish conformation from configuration.Configuration denotes the geometric possibilities for a particular set of atoms. In changing configuration, covalent bonds must be broken. A particular stereochemistry about a given center is consider
32、ed to be a configuration. The primary sequence of a protein is a configuration.Conformation denotes the 3-D architecture of a protein. It is established by a variety of weak forces. In contrast to configuration,a conformation can change readily. The conformation of a protein is first and foremost es
33、tablished by its primary structure (amino acid sequence). Its interaction with solvent (generally H2O) and the pH and ionic composition are also critical in establishing and/or maintaining a proteins conformation. How to investigate protein tertiary structure?X- ray crystal diffractionNMR ( less tha
34、n 120aa)Forces that Influence Protein Tertiary Structure Hydrogen bonds - The atoms of the peptide bond will tend to form hydrogen bonds whenever possible. Amino acid side chains that are capable of forming hydrogen bonds will typically be found on the surface of proteins, so that they may interact
35、with water. Although the energy of the hydrogen bond (12 kJ/mol) is fairly weak when compared to covalent interactions, they are numerous, and together contribute a significant amount of energy and stability to protein conformation. Hydrophobic interactions - The interior of are proteins almost excl
36、usively contain amino acids with hydrophobic side chains. Well find that the need to bury hydrophobic side chains of amino acids is what drives a protein to fold into its proper conformation. Van der Waals interactions - induced electrical interactions. Contribute significantly to conformational sta
37、bility in the interior of the protein. Electrostatic Interactions Disulfide bondMotifsMotif is a simple combination of a few secondary structural elements with a specific geometric arrangement.Some motifs are associated with a particular biological function while others are not but rather are part o
38、f a larger structural assembly. Length of loop isgenerally two to five residuesb b-hairpinFound in almost every protein that has a parallel b b-sheetThe Helix-Turn-Helix Motif1.Involved in DNA recognition2.Found in prokaryotic systems3.Typically less than 100 amino acids4.In eukaryotic systems, ther
39、e is the homeodomain which is involved in DNA binding5.In eukaryotic systems, there are other motifs - zinc fingers and leucine zippers6.First observed in CAP in Escherichia coliCro repressor66 amino acidsl l repressor/DNA complexThe EF Hand1.It is a helix-loop-helix motif but with a completely diff
40、erent disposition of the -helices and the actual types of amino acids present.2.This type of motif was first observed in parvalbumin which is a muscle protein from carp.3.The calcium ion is coordinated by seven oxygens. ParvalbuminThe Immunoglobulin FoldIt is a compressed anti-parallel b b-barrel bu
41、ilt up from one three-stranded b b-sheet packed against a four-stranded anti-parallel sheet. It contains a Greek key in its fold.The b b-helix1.first observed in pectate lyase C of Erwinia chrysanthemi in 19932.has now been observed in more than a dozen proteins - some are enzymes and some are not -
42、 for example, the filamentous hemagglutinin secretion domain contains this motif (PNAS, Vol 101, p. 6194, April 2004). 3.the structural motif consists of three parallel b b -sheets that wrap around to form a b b-helix. Protein DomainsProtein domains are distinct, stable, globular units or folds with
43、in a single polypeptide.Frequently domains will retain their correct three-dimensional structure when cleaved proteolytically from the parent peptide, or when expressed as a singly entity.Different domains will often have distinct functions in the parent protein.The muscle protein, troponin C (shown
44、 at left), appears like a dumbell. The two lobes (blue and purple), which represent two domains, function to bind calcium ions.DNA polymerase has three domains A domain is a segment of the polypeptide that forms a stable structure independently of the the remainder of the protein.Quaternary Structur
45、eThe majority of proteins in nature exist as oligomers, which are complexes (noncovalent associations) of two or more monomer subunits. The number and arrangement of these complexes is referred to the proteins quaternary structure. As previously discussed, these subunits can be identical (homomultim
46、ers), or nonidentical (heteromultimers). Hemoglobin is composed of four chains, which are two alpha chains and 2 beta chains. It is a tetramer, or a dimer of b protomers.A protomer is the simplest repeating structural unit of an oligomeric protein.The advantages of oligomerization are multifold. 1.
47、There is usually an increase in stability of the protein, due to a less surface to volume ratio. 2. Allows for regulation. 3. Less DNA is need to encode a homomultimer than a large polypeptide of a similar size. 4o structure: hemoglobin is composed of four polypeptide chains A cartoon representation
48、 of deoxyhemoglobin is shown (the hemes are not shown). The quaternary structure of a protein refers to how the polypeptide chains (also known as the protein subunits) pack together.Sickle cell disease: The Glu to Val mutation alters the surface charge in hemoglobin S A spacefilling model of hemoglo
49、bin is shown. Heme groups are shown in black and Glu 6 is shown in red. Mutation of Glu 6 to Val in hemoglobin S results in a hydrophobic patch on the surface of the protein. In sickle cell disease, deoxyhemoglobin S forms 14-stranded helices which are insoluble and lead to formation of the sickle-s
50、haped red cell. A model of the 14-stranded helix is shown at the left.SummaryProteins have four levels of structural organization Primary structure = linear sequence of amino acids Secondary structure = -helices and b b-sheets Tertiary structure = packing of 2o structures Quaternary structure = pack
