lipid

Molecular Biochemistry I

Lipids and Membrane Structure

Contents of this page:
Fatty acids
Glycerophospholipids
Sphingolipids
Bilayer membranes & membrane fluidity
Cholesterol
Lateral mobility & flip flop
Lipid rafts & caveolae
Membrane proteins
Integral protein structure
Lipids are non-polar (hydrophobic) compounds, soluble in organic solvents.
Most membrane lipids are amphipathic, having a non-polar end and a polar end.

Fatty acids consist of a hydrocarbon chain with a carboxylic acid at one end. A 16-carbon fatty acid is represented at right and below.

CH3(CH2)14-COO- Non-polar__  polar

A16-carbon fatty acid, with one cis double bond between carbon atoms 9 and 10 may be represented as 16:1 cisD9. A diagram of this fatty acid is at right. Double bonds in fatty acids usually have the cis configuration. Most naturally occurring fatty acids have an even number of carbon atoms. Examples of fatty acids with their common names are listed below. See also the table on p. 383 of Voet & Voet, Biochemistry, 3rd Edition.

14:0	myristic acid
16:0	palmitic acid
18:0	stearic acid
18:1 cisD9	oleic acid
18:2 cisD9,12	linoleic acid
18:3 cisD9,12,15	linonenic acid
20:4 cisD5,8,11,14	arachidonic acid
20:5 cisD5,8,11,14,17	eicosapentaenoic acid (an omega-3 fatty acid because of double bond 3 C from distal end)

There is free rotation about C-C bonds in the fatty acid hydrocarbon, except where there is a double bond. Each cis double bond causes a kink in the chain, as emphasized in the diagram above. Rotation about other C-C bonds would permit a more linear structure than is shown, but there would be a kink.

Glycerophospholipids (phosphoglycerides) are common constituents of cellular membranes. They have a glycerol backbone. The hydroxyls at C1 & C2 of glycerol are esterified to fatty acids.

Recall that an ester linkage forms when a hydroxyl reacts with a carboxylic acid, with loss of water.

In phosphatidate, fatty acids are esterified to the hydroxyls on C1 and C2, while the C3 hydroxyl is esterified to phosphate.

In most glycerophospholipids (phosphoglycerides), the phosphate is in turn esterified to an alcohol of one of the following polar head groups: serine, choline, ethanolamine, glycerol, or inositol (designated X  at right; see structures in Voet & Voet p. 385). Color is also used to distinguish the fatty acids,  glycerol, and phosphate.  The two fatty acids tend to be non-identical. They may differ in length and/or the presence or absence of double bonds.

Phosphatidylinositol, with inositol as polar head group, is one glycerophospholipid. In addition to being a membrane lipid, phosphatidylinositol has roles in cell signaling, to be discussed later.

Phosphatidylcholine, with choline as polar head group, is another glycerophospholipid. It is a common membrane lipid.

Each glycerophospholipid has: a polar region [glycerol, carbonyl oxygen atoms of fatty acids, phosphate, and the polar head group (designated X above)] non-polar  hydrocarbon tails of fatty acids (designated R1, R2 above).  Explore below right the glycerophospholipid dioleoyl-phosphatidylcholine.

The data are from a crystal structure of the lipid bound to a protein, PDB 1T27, solved by M. D. Yoder, L. M. Thomas, J. M. Tremblay, R. L. Oliver, L. R. Yarbrough & G. M. Helmkamp in 2001. Display as ball & stick. Note that H atoms are not visible in this X-ray crystal structure. Using the diagram of phosphatidylcholine above, and the color code given, identify each of the following:    glycerol backbone    phosphate    choline    fatty acids (oleic acids) Oleic acid has a double bond between C atoms # 9 & 10 (count from the carbonyl C). Identify atoms involved in the double bond, and note how bond angles differ at this location. (Look for the "kink" in each fatty acid.) You may wish to change the display to sticks.

Dioleoyl-phosphatidylcholine    C   O   N   P

Sphingolipids are derivatives of the lipid sphingosine. Sphingosine has a long hydrocarbon tail, and a polar domain that includes an amino group. Sphingosine may be reversibly phosphorylated to produce the signal molecule sphingosine-1-phosphate. Other derivatives of sphingosine are commonly found as constituents of biological membranes.

The amino group of sphingosine can form an amide bond with a  fatty acid carboxyl, to yield a ceramide. In the more complex sphingolipids, a polar "head group" is esterified to the terminal hydroxyl of the sphingosine moiety of the ceramide.

Sphingomyelin has a phosphocholine or phosphoethanolamine head group. Sphingomyelins are common constituents of plasma membranes. Sphingomyelin, with a phosphocholine head group, is comparable in size and shape to the glycerophospholipid phosphatidyl choline. (Figs 12-4 & 12-6, p. 386, 387).

A cerebroside is a sphingolipid (ceramide) with a monosaccharide such as glucose or galactose as polar head group.A ganglioside is a ceramide with a polar head group that is a complex oligosaccharide, including the acidic sugar derivative sialic acid. See p. 388. Cerebrosides and gangliosides, which are collectively called glycosphingolipids, are commonly found in the outer leaflet of the plasma membrane bilayer, with their sugar chains extending out from the cell surface.

Amphipathic lipids in association with water form complexes in which their polar regions are in contact with water and their hydrophobic regions are away from water. Depending on the lipid, possible molecular arrangements include (p. 390-391):

Various micelle structures. E.g., the spherical micelle is a stable configuration for amphipathic lipids that have a conical shape, such as fatty acids. A bilayer. This is the most stable configuration for amphipathic lipids with a cylindrical shape, such as phospholipids.

Membrane fluidity: The interior of a lipid bilayer is normally highly fluid (discussed p. 393-394). In the liquid crystal state, hydrocarbon chains of phospholipids are disordered and in constant motion. At lower temperature, a membrane containing a single phospholipid type undergoes transition to a crystalline state in which fatty acid tails are fully extended, packing is highly ordered, and van der Waals interactions between adjacent chains are maximal. Kinks in fatty acid chains, due to cis double bonds, interfere with packing of lipids in the crystalline state, and lower the phase transition temperature.

Cholesterol, an important constituent of cell membranes, has a rigid ring system and a short branched hydrocarbon tail. Cholesterol is largely hydrophobic. But it has one polar group, a hydroxyl, making it amphipathic. See also p. 388-389.

Cholesterol viewed by Chime, in sticks & spacefill displays.

Cholesterol synthesis and transport are discussed elsewhere.Cholesterol inserts into bilayer membranes with its hydroxyl group oriented toward the aqueous phase and its hydrophobic ring system adjacent to fatty acid tails of phospholipids. The hydroxyl group of cholesterol forms hydrogen bonds with polar phospholipid head groups.

Interaction with the relatively rigid cholesterol decreases the mobility of hydrocarbon tails of phospholipids. But the presence of cholesterol in a phospholipid membrane interferes with close packing of fatty acid tails in the crystal state, and thus inhibits transition to the crystalline state. Phospholipid membranes with a high concentration of cholesterol have a fluidity intermediate between the liquid crystal and crystal states.

Two strategies by which phase changes of membrane lipids are avoided: Cholesterol is abundant in membranes, such as plasma membranes, that include many lipids with long-chain saturated fatty acids. In the absence of cholesterol, such membranes would crystallize at physiological temperatures. The inner mitochondrial membrane lacks cholesterol, but includes many phospholipids whose fatty acids have one or more double bonds, which lower the melting point to below physiological temperature.

Lateral mobility of a lipid, within the plane of a membrane, is depicted at right and in the animation provided.  High speed tracking of individual lipid molecules has shown that lateral movements are constrained within small membrane domains. Hopping from one domain to another occurs less frequently than rapid movements within a domain. The apparent constraints on lateral movements of lipids (and proteins) has been attributed to integral membrane proteins, anchored  to the cytoskeleton, functioning as a picket fence. For more information, see slide shows in a website of the Kusumi laboratory.

of lipid lateral diffusion

Flip-flop of lipids (from one half of a bilayer to the other) is normally very slow. Flip-flop would require the polar head-group of a lipid to traverse the hydrophobic core of the membrane. The two leaflets of a bilayer membrane tend to differ in their lipid composition (see p. 406). Flippases catalyze flip-flop in membranes where lipid synthesis occurs, and some membranes contain enzymes that actively transport particular lipids from one monolayer to the other.

of lipid flip-flop

Membrane Proteins may be classified as peripheral, integral, or having a lipid anchor.Peripheral proteins are on the membrane surface. They are water-soluble, with mostly hydrophilic surfaces. Often peripheral proteins can be dislodged from membranes by conditions that disrupt ionic and H-bond interactions, e.g., extraction with solutions containing high concentrations of salts, change of pH, and/or agents (chelators) that bind divalent cations.

Many proteins have a modular design, with different segments of the primary structure folding into domains with different functions.

Some cytosolic proteins have domains that bind to polar head groups of lipids that transiently exist in a membrane. The enzymes that create or degrade these lipids are subject to signal-mediated regulation, providing a mechanism for modulating affinity of a protein for a membrane surface. For example, pleckstrin homology (PH) domains bind to phosphorylated derivatives of phosphatidylinositol (PI). Some pleckstrin homology domains bind to PIP2 (PI-4,5-P2), shown at right.

Other pleckstrin homology domains recognize and bind to phosphatidylinositol derivatives with Pi esterified at the 3' hydroxyl of inositol. Examples include PI-3-P (at right), PI-3,4-P2, and PI-3,4,5-P3.

Lipid anchor: Some proteins bind to membranes via a covalently attached lipid anchor, that inserts into the bilayer (see p. 402-404). A protein may link to the cytosolic surface of the plasma membrane via a covalently attached fatty acid (e.g., palmitate or myristate) or an isoprenoid group. Palmitate is usually attached via an ester linkage to the thiol of a cysteine residue, as shown at right. A protein may be released from the plasma membrane to the cytosol via depalmitoylation, hydrolysis of the ester linkage.

An isoprenoid group, such as a farnesyl residue, is attached to some proteins via a thioether linkage to a cysteine thiol, as shown at right.

Glycosylphosphatidylinositols, abbreviated GPI, are complex glycolipids that attach some proteins to the outer surface of the plasma membrane (see p. 404). The linkage is similar to the following, although the oligosaccharide composition may vary:

protein (C-terminus) - phosphoethanolamine - mannose - mannose - mannose - N-acetylglucosamine - inositol (of membrane-embedded phosphatidylinositol)

The protein is tethered some distance out from the membrane surface by the long oligosaccharide chain. GPI-linked proteins may be released from the outer cell surface by phospholipases.
Integral proteins have domains that extend into the hydrocarbon core of the membrane. Often they span the bilayer. Intramembrane domains have largely hydrophobic surfaces, that interact with membrane lipids. See Fig. 12-18 p. 395.

Amphipathic detergents are required for solubilization of integral proteins. Hydrophobic domains of detergents substitute for lipids in coating hydrophobic protein surfaces. Polar domains of detergents interact with water. If detergents are removed, purified integral proteins tend to aggregate and come out of solution. Their hydrophobic surfaces associate to minimize contact with water.

Lipid rafts: Complex sphingolipids tend to separate out from glycerophospholipids and co-localize with cholesterol in membrane microdomains called lipid rafts. Membrane fragments assumed to be lipid rafts are found to be resistant to detergent solubilization, which has facilitated their isolation and characterization. Differences in molecular shape may contribute to the tendency for sphingolipids to separate out from glycerophospholipids in membrane microdomains. Sphingolipids usually lack double bonds in their fatty acid chains. In contrast, glycerophospholipids often include at least one fatty acid that is kinked, due to one or more double bonds. See an online diagram from an article by J. Santini & coworkers. Hydrogen bonding between the hydroxyl group of cholesterol and the amide group of sphingomyelin may in part account for the observed affinity of cholesterol for sphingomyelin in raft domains. Lipid raft domains tend to be thicker than adjacent membrane areas, in part because the saturated hydrocarbon chains of sphingolipids are more extended. Proteins involved in cell signaling often associate with lipid raft domains. Otherwise soluble signal proteins often assemble in complexes at the cytosolic surface of the plasma membrane in part via insertion of attached fatty acyl or isoprenoid lipid anchors into raft domains.  Integral proteins may concentrate in raft domains via interactions with raft lipids or with other raft proteins. Some raft domains contain derivatives of phosphatidylinositol that bind signal proteins with pleckstrin homology domains. Caveolae are invaginated lipid raft domains of the plasma membrane that have roles in cell signaling and membrane internalization. Caveolin is a protein associated with the cytosolic leaflet of the plasma membrane in caveolae. Caveolin interacts with cholesterol and self-associates as oligomers that may contribute to deforming the membrane to create the unique morphology of caveolae. Additional websites of interest: Electron micrograph and information about caveolae (home page of Deborah Brown at SUNY Stony Brook). Diagram and information about lipid rafts (website maintained by the Maciver lab at University of Edinburgh).

Integral protein structure Atomic-resolution structures have been determined for only a small number of integral membrane proteins. Integral proteins are difficult to crystallize for X-ray analysis. Because of their hydrophobic transmembrane domains, detergents must be present during crystallization. A membrane-spanning a-helix is the most common structural motif found in integral proteins.

In an a-helix, amino acid R-groups protrude out from the helically coiled polypeptide backbone. The largely hydrophobic R-groups of a membrane-spanning a-helix contact the hydrophobic membrane core, while the more polar peptide backbone is buried. In the images at right, H atoms are not visible. Colors: C  N  O  R-group

Particular amino acids tend to occur at different positions relative to the surface or interior of the bilayer in transmembrane segments of integral proteins has shown that . Residues with aliphatic side-chains (leucine, isoleucine, alanine, valine) predominate in the middle of the bilayer.

Tyrosine and tryptophan are common near the membrane surface. It has been suggested that the polar character of the tryptophan amide group and the tyrosine hydroxyl, along with their hydrophobic ring structures, suit them for localization at the polar/apolar interface. Lysine and arginine are often at the lipid/water interface, with the positively charged groups at the ends of their aliphatic side chains extending toward the polar membrane surface.

Cytochrome oxidase is an example of an integral protein whose intramembrane domains consist mainly of transmembrane a-helices. For another example, see notes on the protein rhodopsin. Explore below the transmembrane a-helix colored green at the far left in this ribbon display of cytochrome oxidase.

Display as sticks. Rotate to view the a-helix from the side & down its axis. Identify the polar backbone atoms using CPK color. To distinguish the amino acid side-chains (R groups), select protein, sidechain and then change the display, e.g., to ball & stick. What part of such a transmembrane a-helix would mainly contact the lipid core of a membrane? Identify each amino acid. Examples of some amino acid structures are shown below. Questions to be answered: 1. What is the location of the partly polar/partly hydrophobic residues tyrosine and tryptophan? 2. What is the location and orientation of a lysine residue and it's side-chain amino group? What is the significance of this location and orientation? 3. What types of amino acids are in the part of the transmembrane a-helix corresponding to the middle of the membrane? Why might this be? A few amino acid structures:

C   O   N   S

Hydropathy plots: A 20-amino acid a-helix just spans a lipid bilayer.  Hydropathy plots are used to search for 20-amino acid stretches of hydrophobic residues in the primary sequence of a protein for which a crystal structure is not available (Fig. 12-22 p. 397). Putative hydrophobic transmembrane a-helices have been identified this way in many membrane proteins. It should be emphasized that hydropathy plots alone are not conclusive. Protein topology studies are used to test the transmembrane distribution of protein domains predicted by hydropathy plots. E.g., if a hydropathy plot indicates one 20-amino acid hydrophobic stretch (one putative transmembrane a-helix), topology studies are expected to confirm location of N and C termini on opposite sides of the membrane. If two transmembrane a-helices are predicted, N and C termini should be on the same side of the membrane. The segment between the transmembrane a-helices should be on the other side of the membrane.

Transmembrane topology is tested with impermeant probes, added on one side of a membrane. For example:

• Protease enzymes. Degradation of a protein segment would indicate exposure to the aqueous phase on the side of the membrane to which the protease was added.
• Monoclonal antibodies raised to peptides equivalent to individual segments of the protein. Binding would indicate surface exposure of that protein segment on the side to which Ab was added.

Transmembrane topology studies have shown that all copies of a given type of integral protein have the same orientation relative to the bilayer membrane. Flip-flop of integral proteins does not occur.
Do a hydropathy plot (studio exercise).

A helical wheel diagram looks down the axis of an a helix, projecting amino acid side-chains onto a plane (see Fig. 8-43 p. 247). Transmembrane a-helices that line a water-filled channel might have polar amino acid R-groups (side-chains) facing the lumen, and non-polar amino acid R-groups facing lipids or other hydrophobic a-helices. Such mixed polarity would prevent detection by a hydropathy plot.

While transmembrane a-helices are the most common structural motif for integral proteins, a family of bacterial outer envelope channel proteins called porins have instead b barrel structures. A b barrel is a b-sheet rolled up to form a cylindrical pore. At right is shown one channel of a trimeric porin complex.

In a b-sheet, amino acid R-groups alternately point above and below the sheet. See simplified cartoon at right and diagram p. 228. Much of the primary structure of a porin consists of alternating polar and non-polar amino acids. Polar residues face the aqueous lumen. Non-polar residues are in contact with membrane lipids.

Explore below a sucrose-selective porin from Salmonella typhimurium. (PDB 1AOS, structure solved by D. Forst, W. Welte, T. Wacker & K. Diederichs in 1998.)

Color chain and display as cartoons. Drag to view channel structures. Now select protein, hydrophobic and then select, change color to and specify a color. Do the same for polar residues. In addition to viewing in cartoon mode, select protein, protein and change the display to spacefill. Note the location of polar & non-polar residues. Record the sequence of amino acids in one of the outer b-strands. You might want to try this in backbone display after selecting and hiding two of the chains. Do polar and non-polar residues alternate? Look for particular amino acids (e.g., Lys, Trp, Tyr, Leu, etc.) among residues facing outward toward the membrane, either near the membrane surface or in the middle of the bilayer. Can you find examples of amino acid location similar to what you observed for a transmembrane a-helix?

amino acid & peptides

Amino Acids and Peptides

Lecture Notes | 462a Home Reading - Chapter 5 Practice problems - Chapter 5 - 1-5, 7, 8, 10 (For #10, use "generic" pK values for groups in peptides and proteins from THESE LECTURE NOTES, not values for free amino acids from text table; that of course means different answers from back of book!); Amino Acids and Peptides extra problems [PDF] Acid dissociation reactions for functional groups of amino acid residues in proteins: [PDF] Key Concepts Properties of the 20 amino acids that occur in peptides and proteins are crucial to the structure and function of proteins. stereochemistry relative hydrophobicity or polarity hydrogen bonding properties ionization properties other chemical properties Condensation of 2 amino acids forms the peptide bond, the amide linkage holding amino acid residues in peptide and protein polymers. Properties of the peptide bond have major consequences in terms of the 3-dimensional structures of proteins There's an excellent website on amino acids being developed here in the Department of Biochemistry and Molecular Biophysics; parts of it are still under construction, but there are links to various very useful parts of it here in these notes, and indeed parts of it may be used in class. BASICS Proteins are polymers of a-amino acids:  There are 20 different amino acids found in proteins and they differ by the nature of the R group.  Both the a-amino group (amino group substituent on the aC) and the a-carboxyl group (carboxyl substituent on the aC) are ionizable. a-COOH group:  a weak acid, can DONATE its proton, with a pKa of about 2-3. What's the conjugate base form of the carboxyl group? Which form is charged, and is it a positive or a negative charge? a-NH2 group:  a weak base (there's an unshared pair of electrons on the N; the neutral amino group can ACCEPT a proton). What's the conjugate acid form of the amino group? Which form is charged, and is it a positive or a negative charge? pKas of a-amino and a-carboxyl groups are different for different amino acids, and also are altered if they're the terminal groups on a chain of amino acids, i.e., a peptide or protein. The nonionic form shown above does NOT occur in water. WHY NOT? Predominant form in H2O is the zwitterion:   .   Stereochemistry of the amino acids a-carbon is asymmetric (has four different substituents) except for one amino acid, for which the R group is a hydrogen atom. amino acids occur as enantiomers (nonsuperimposable complete mirror images) L-amino acids are the naturally occurring enantiomers found in all proteins There are naturally occurring D-amino acids, but not in proteins (found in some bacterial cell wall peptide structures, in some peptide antibiotics, etc.) (D_L) Perspective formulas show stereochemistry; projection formulas CAN be written "correctly", with convention that horizontal bonds project out of paper and vertical bonds behind plane of paper, but often biochemists use projection formulas casually (inaccurately), knowing that if it's in a protein, it's always an L-amino acid. (See also Fig. 5-3 in Lehninger Principles.) Fig. 5-4 (Nelson & Cox: Lehninger Principles of Biochemistry): Absolute configurations of D-glyceraldehyde as the reference compound for a-amino acids.  D- and L- apply only to the absolute configuration around the chiral a carbon; 2 of the 20 amino acids (threonine and isoleucine) have a second chiral center, requiring the RS system to describe their structures accurately, but we aren't going to worry about using the RS system here. Which of the amino acids does NOT have a chiral center, so has no D/L isomers? Amino Acid Abbreviations amino acid (or residue in protein) 3-letter abbreviation 1-letter abbreviation Mnemonic for 1-letter abbreviation Glycine Gly G Glycine Alanine Ala A Alanine Valine Val V Valine Leucine Leu L Leucine Isoleucine Ile I Isoleucine Proline Pro P Proline Methionine Met M Methionine Phenylalanine Phe F Fenylalanine Tryptophan Trp W tWyptophan (or tWo rings) Tyrosine Tyr Y tYrosine Serine Ser S Serine Threonine Thr T Threonine Cysteine Cys C Cysteine Aspartic Acid Asp** D asparDic acid Glutamic Acid Glu* E gluEtamic acid Asparagine Asn** N asparagiNe Glutamine Gln* Q Q-tamine Histidine His H Histidine Lysine Lys K (before L) Arginine Arg R aRginine * Glx = either acid or amide (when it isn't known which it is) **Asx = either acid or amide (when it isn't known which it is) Properties of Amino Acid Side Chains Side chains ("R groups") provide proteins with unique structural and functional properties. Additional C atoms in R groups (after the a C) designated by successive Greek letters: b, g, d, e, as shown in the structure of the amino acid LYSINE (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., p. 116): Side chain classes The side chains of the amino acids play an essential role in determining the properties of proteins.  There is a wide diversity in the chemical properties of amino acid side chains, but they can be grouped into classes, sometimes with overlapping "membership" (e.g., tyrosine is both aromatic and hydroxyl-containing). Other classifications are also possible (for example, the 5 classes in textbook, Fig. 5-5, discussed below).  You are expected to know all 20 amino acid structures and their R group properties, including ionization properties (see table below with "generic" pKa values for groups in peptides and proteins and links to titration curves, and the PDF of proton dissociation reactions). Side Chain Class Amino Acids Aliphatic glycine, alanine, valine, leucine, isoleucine Cyclic proline Aromatic phenylalanine, tyrosine, tryptophan Hydroxyl-Containing serine, threonine, tyrosine Sulfur-Containing cysteine, methionine Basic histidine, lysine, arginine Acidic and Their Amides aspartic acid, glutamic acid, asparagine, glutamine Structures and classification below are from Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., Fig. 5-5. States of ionization are the PREDOMINANT forms found at pH 7. Nonpolar, aliphatic R groups Gly: quite water-soluble (as is Pro) Ala, Val , Leu and Ile: increasing hydrophobicity with increasing number of C atoms in hydrocarbon chain Pro: cyclic (--> unusual properties) shares many properties with the aliphatic group rigidity of ring plays critical role in protein structure (more about that later) Met: methyl thioether (S-containing) quite hydrophobic Met's terminal methyl group important in metabolism Aromatic R groups Phe: phenyl group (linked to b-CH2, so Phe = alanine with a phenyl substituent on the methylene C) VERY hydrophobic. Trp: indole functional group on bC electronegative atom in ring system not as hydrophobic as Phe hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Tyr: phenylalanine with aromatic OH group (phenolic OH) = p-hydroxyphenylalanine ionizable (pKa around 10; loss of proton gives phenolate anion) hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Tyr R group is the least hydrophobic of the 3 aromatic amino acid side chains. Polar, uncharged R groups Ser and Thr: aliphatic OH groups, not ionizable in pH range 1-13 pKa values so high that under any biologically reasonable pH conditions they're polar but not ionizable. hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Asn and Gln: amide functional groups VERY polar, but NOT ionizable hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Cys: thiol (also called a sulfhydryl group) -- not very polar, and IS ionizable sulfur atom makes protonated -SH group more hydrophobic than an aliphatic OH group thiol DOES lose its proton in physiologically relevant pH range (pKa about 8.5) generates -S- (thiolate anion is quite hydrophilic due to the charge). Positively charged R groups (sometimes called "basic" R groups) Arg: guanidino group VERY high pKa (~12+), so a very weak acid (stronger base) carries + charge all across physiological pH range resonance forms of guanidino group stabilize protonated form (charge is delocalized) hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Lys: e-amino group (a primary amine) pKa about 10 protonated form (predominates at physiological pH) carries + charge hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) His: imidazole functional group (has 2 N atoms in 5-membered unsaturated ring) pKa about 6-6.5 protonated form carries + charge, but at pH 7 predominant form is neutral (despite textbook's categorization as "positively charged") very important player in catalytic activity of many enzymes hydrogen bonding capability, and also proton donor/acceptor Negatively charged R groups (sometimes called "acidic" R groups) Asp and Glu: side chain carboxyl groups pKa values around 4 predominant form at physiological pH = carboxylate anion hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?) Relative hydrophobicity/hydrophilicity of amino acid R groups Table 12.2 (Berg, Timoczko and Stryer, Biochemistry, 5th ed.): Polarity scale for amino acid residues based on free energy changes for moving a residue from a hydrophobic environment (dielectric constant = 2) into H2O. Similar trends for relative hydrophobicities in text Table 5-1 (diff. numerical scale, and not arranged in order of relative polarity) Depending on how transfer experiments are done, different absolute numbers can be obtained, but the general trends of relative polarity are clear Phe, Met, Ile, Leu, Val are very hydrophobic Arg, Asp, Lys, Glu, Asn, Gln, and His are quite hydrophilic The rest are in between -- neither very polar nor very hydrophobic Fig. 5-7: Reversible oxidation of 2 cysteine side chain thiols to form cystine, or re-reduction to 2 thiols disulfide bonds between 2 Cys residues in a (usually extracellular) protein often a critical structural feature in extracellular proteins (stabilize folded structures, in interior of protein structure) When found in intracellular proteins, usually have a functional role. Ionization Properties of Amino Acid Functional Groups (in PEPTIDES AND PROTEINS) weak conjugate acid/base groups in peptides and proteins crucial to functions only one a-amino and one a-carboxyl group on a peptide or proteins (at the termini of the chain) because the rest of the a-amino and a-carboxyl groups are tied up in amide bonds holding monomers together in polymer (more later) side chain ionizable groups (only 7 of the 20 amino acids) PDF of the acid dissociation reactions for functional groups of amino acid residues in peptides and proteins ionization states of side chain weak acid groups control charges on protein Note: local environment in peptide or protein determines actual pKa of that specific group, so the ranges shown below (and the rather arbitrary "generic" values, rounded off for simplicity) are only the usual expected ranges for pKa values for the functional groups in peptides and proteins; the pKa of a specific group in a specific protein can lie significantly outside the expected range if the local environment is unusual. links in table below are to titration curves for that amino acid or functional group Group usual pKa range, in peptides & proteins (approx."generic"pKa ) a-Carboxyl (terminal group of peptide or protein) ~3.0 - 4.0 (generic 3.0) Asp, Glu (side chain carboxyl) ~4.0 - 4.5 (generic 4.0) His (imidazole) ~6.0 - 7.4 (generic 6.5) Cys (thiol, SH) ~8.5 - 9.0 (generic 8.5) Tyr (phenolic OH) ~9.5 - 10.5 (generic 10.0) a-Amino (terminal group of peptide or protein) ~8.0 - 9.0 (generic 8.0) Lys (e-amino) ~9.8 - 10.4 (generic 10.0) Arg (guanidino) ~12.0 - 12.5 (generic 12.0) Isoelectric point (pI) pI = "isoelectric pH" = "isoelectric point" = pH at which the NET charge on a molecule is ZERO.  If pH < pI, net charge is positive (more + than - charges) If pH > pI, net charge is negative (more - than + charges) pI = the pH exactly halfway between the two pKa values surrounding the zero net charge equivalence point on the titration curve (examples to be analyzed in class: Gly and His) Fig. 5-10. Titration curve of glycine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.) Fig. 5-12b. Titration curve of histidine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.) Molecular separations based on charge properties (paper electrophoresis of amino acids as an example) paper strip soaked in buffer, in contact with 2 reservoirs with electrodes connected to a power supply Buffer reservoir #1 + (anode; anions move toward it) O Buffer reservoir #2 _ (cathode; cations move toward it) ^ origin (sample of an amino acid applied) When a voltage is applied, in which direction will the amino acid move? What do you need to know to answer that question? ________________________ ________________________ e.g., Histidine: (sample dissolved in buffer, applied at origin above, and voltage applied) > buffer pH net charge direction of migration 1 3.9 7.6 11 Ultraviolet absorbance of amino acid side chains Aromatic amino acids (Trp, Tyr, Phe) absorb light in the near ultraviolet region of the spectrum (250-300 nm).  Trp has highest molar absorptivity, followed by Tyr, with Phe making only a small contribution. Disulfide bonds (between Cys residues in proteins) also absorb in the uv range, but much less than the aromatics. Fig. 5-6 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Absorbance of ultraviolet light by aromatic amino acids Posttranslational modifications of amino acid side chains chemical modifications AFTER biosynthesis of proteins occur for a few amino acid residues in some proteins Some examples (see also Fig. 5-8, Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)): O-Phosphoserine 4-Hydroxyproline 5-Hydroxylysine g-carboxyglutamate reversible phosphorylation and dephosphorylation of Ser, Thr, and Tyr residues very important in covalent regulation of activity of some enzymes and many biosignalling proteins, including some hormone receptors and transcription factors 4-hydroxyproline & 5-hydroxylysine important in structure of collagen (fibrous protein in connective tissue) g-carboxyglutamate important in a number of proteins whose function involves Ca2+ binding, including several proteins involved in blood clotting Chemical Reactions of Amino Acids All amino acids have at least two reactive groups: the a-amino and a-carboxyl groups and these groups can react with a variety of reagents. Here are two examples: A particularly interesting example is the green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria, which has generated intense interest as a marker for gene expression and localization of gene products.  The chromophore, which results from the spontaneous cyclization and oxidation of the sequence -Ser65-Tyr66-Gly67- , is unusual because it does not involve a non-protein chromophore, as is usually the case for colored proteins. The chromophore is buried in the interior of GFP. The Peptide Bond Peptides and proteins:polymers of amino acids joined bypeptide bonds amide linkages from condensation of a-carboxyl group of one amino acid with a-amino group of another amino acid process repeated many times --> linear chain of amino acids, a polypeptide chain convention: sequence written from left to right starting with residue with free a-amino group (the N-terminal or amino terminal amino acid residue) and ending with the residue containing the free a-carboxyl group (the C-terminal or carboxyl terminal residue), e.g., NH2-Glu-Gly-Ala-Lys-COOH = EGAK average residue mass ~110 (average Mr of the 20 amino acids minus Mr of H2O) a polypeptide chain with 100 amino acid residues would have a Mr of about 11,000) small peptides (a "few" amino acid residues) = oligopeptides Peptide bond formation endergonic (DGo' ~21 kJ/mol) (How would a cell make the reaction go in the direction of condensation in an aqueous environment? no details needed here for biochemical mechanism -- that's covered in BIOC 411) peptide bonds metastable in aqueous environment -- equilibrium lies far in direction of hydrolysis, but RATE of hydrolysis very slow in absence of catalyst Enzymes that catalyze peptide bond hydrolysis = peptidases or proteases, e.g., (specific examples of proteases) your digestive proteases like trypsin and pepsin Ionization properties of peptides analyzed the same way as for free amino acids one a-amino group (pKa approx. 8) and one a-carboxyl group (pKa approx. 3), plus any ionizable side chains on residues in the peptide To figure out approximate net charge of a peptide at a given pH: make yourself notes on the sequence to keep track of what you're doing add up charges on all the ionizable groups Example: Fig. 5-14 (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.): pentapeptide SGYAL = Ser-Gly-Tyr-Ala-Leu = Serylglycyltyrosylalanylleucine Amino Acid Analysis Sequence of amino acids in a protein is dictated by the sequence of nucleotides in the gene encoding that protein: (from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., p. 28) Each protein (unique sequence) has unique amino acid composition. Can chemically hydrolyze (hot 6N HCl) a pure protein to generate the free amino acids and determine its amino acid composition chromatographically Because side chains of the amino acids have different properties, can separate and quantitate all 20 amino acids using a variety of chromatographic techniques, as illustrated below. Peptide bond has resonance structures --> partial double bond character Due to the partial double bond character of the peptide bond, the O, C, N and H atoms are nearly planar and there is no rotation about the peptide bond (peptide).  As we shall see later, the planarity of the these elements has important consequences for the three dimensional structure of proteins.  Generally, the two Ca groups are in a trans configuration, which minimizes steric interaction (cis/trans).