Reginald H. Garrett Charles M. Grisham Reginald Garrett & Charles Grisham University of Virginia Chapter 5 Proteins: Their Primary Structure and Biological Function

Essential Question What structural forms do polypeptide chains assume, how can the sequence of amino acids in a protein be determined, and what are the biological roles played by proteins?

Outline What is the fundamental structural pattern in proteins? What architectural arrangements characterize protein structure? How are proteins isolated and purified from cells? How is the amino acid analysis of proteins performed? How is the primary structure of a protein determined? Can polypeptides be synthesized in the laboratory? What is the nature of amino acid sequences? Do proteins have chemical groups other than amino acids? What are the many biological functions of proteins?

5.1 What Architectural Arrangements Characterize Protein Structure? Proteins are classed according to shape and and solubility Shape - globular or fibrous The four levels of protein structure are: - Primary (1°) - sequence - Secondary (2°) - local structures - H-bonds - Tertiary (3°) - overall 3-dimensional shape - Quaternary (4°) - subunit organization

5.1 What Architectural Arrangements Characterize Protein Structure? (a) Proteins having structural roles in cells are typically fibrous and often water insoluble. (b) Myoglobin is a globular protein. (c) Membrane proteins fold so that hydrophobic amino acid side chains are exposed in their membrane-associated regions.

5.1 What Architectural Arrangements Characterize Protein Structure? Bovine pancreatic ribbonuclease A contains 124 amino acid residues, none of which are Trp. Four disulfide bridges are indicated in gold.

5.1 What Architectural Arrangements Characterize Protein Structure? The α-helix and the β- pleated strand are the two principal secondary structures found in proteins. Secondary structures in proteins

How to view a protein? The tertiary structure of a protein may be viewed in several ways: Backbone only Backbone plus side chains Ribbon structure Space-filling structure Each of these is an abstraction

How to view a protein? Folding of the polypeptide into a compact, roughly spherical conformation creates the tertiary (3°) level of protein structure.

The Quaternary Level of Protein Structure Hemoglobin is a tetramer consisting of two α and two β polypeptide chains.

A Protein’s Conformation Can Be Described as Its Overall Three-Dimensional Structure Be careful to distinguish the terms “conformation” and “configuration” A configuration change require the breaking of a bond. A protein, or any molecule, can change its conformation by changing shape without breaking a bond.

Figure 5.6 Configuration and conformation are not synonymous Imagine the conformational possibilities for a protein in which two of every three bonds along its backbone are freely rotating single bonds.

5.2 How Are Proteins Isolated and Purified from Cells? The thousands of proteins in cells can be separated and purified on the basis of size and electrical charge Proteins tend to be least soluble at their isoelectric point Increasing ionic strength at first increases the solubility of proteins (salting-in), then decreases it (salting-out)

5.2 How Are Proteins Isolated and Purified from Cells? Purification was difficult for a endogenous protein First proteins studies were very abundant Modern cloning techniques all for production of large quantities of specific proteins This process still requires that the protein be isolated from a cell, and purified from the other cellular components

Conditions affect protein Stability pH The wrong pH causes denaturation Temperature The wrong temperature can cause denaturation Presence of other proteins Proteases can destroy proteins Adsorption to surfaces Some proteins can be denatured upon exposure to air Long term storage Most proteins should be stored at -20°C or lower to minimize degradation and denaturation

ELISA Enzyme linked immunosorbent assay Used to determine (quantify) the amount of protein present Protein Concentration

Spectroscopic method for determining protein concentration Beer-Lambert law A= εcl A 280 – absorbance of F, Y, W Protein Concentration

Colorimetric method for determining protein concentration Bradford assay Protein Concentration

Protein Purification

Salting Out

Ion Exchange Chromatography Animation

Gel Filtration Chromatography Animation

Affinity Chromatography Immunoaffinity Metal chelate

5.2 How Are Proteins Isolated and Purified from Cells? A typical protein purification scheme uses a series of separation methods. Note the dramatic increase in specific activity* of the enzyme through a series of five different purification procedures. *The term “specific activity” refers to the activity of the enzyme per mg of protein.

Dialysis Techniques, Figure 1. A dialysis experiment. The solution of macromolecules is placed in a semipermeable membrane bag, and the bag is immersed in a bathing solution. Diffusible solutes in the dialysis bag equilibrate across the membrane.

SDS-PAGE Sodium-dodecyl sulfate – Poly acrylamide gel electrophoresis

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Techniques, Figure 6. A plot of protein mobility versus log of molecular weight of individual peptides.

Two-Dimensional Gel Electrophoresis Techniques, Figure 7. A two-dimensional electrophoresis separation. Macromolecules are first separated according to charge by isoelectric focusing in a tube gel. The gel containing separated molecules is then place on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS- PAGE gel, where they are separated according to size.

Capillary Electrophoresis 2D Gel electrophoresis

Acid hydrolysis liberates the amino acids of a protein Note that some amino acids are partially or completely destroyed by acid hydrolysis Chromatographic methods are used to separate the amino acids The amino acid compositions of different proteins are different 5.3 How Is the Amino Acid Analysis of Proteins Performed?

5.4 How is the Primary Structure of a Protein Determined? The sequence of amino acids in a protein is distinctive Both chemical and enzymatic methodologies are used in protein sequencing

In 1953, Sanger sequenced the two chains of insulin. Sanger's results established that all of the molecules of a given protein have the same sequence. Proteins can be sequenced in two ways: - real amino acid sequencing - sequencing the corresponding DNA in the gene Frederick Sanger was the first to determine the sequence of a protein

The sequence of insulin The hormone insulin consists of two polypeptide chains, A and B, held together by two disulfide (S-S) cross- bridges. The A chain has 21 amino acid residues and an intrachain disulfide; the B polypeptide contains 30 amino acids.

1.If more than one polypeptide chain, the chains are separated and purified. 2.Intrachain S-S (disulfide) cross-bridges are cleaved. 3. The N-terminal and C-terminal residues are identified. 4. Each polypeptide chain is cleaved into smaller fragments, and the composition and sequence of each fragment is determined. 5. Step 4 is repeated, using a different cleavage procedure to generate a different and overlapping set of peptide fragments. 6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments. Determining the Sequence – A Six-Step Strategy

Step 1: Separation of chains Subunit interactions depend on weak forces Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)

Step 2: Cleavage of Disulfide bridges Performic acid oxidation Sulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate

Disulfide cleavage

Step 3: Identify N- and C-terminal residues N-terminal analysis: Edman's reagent phenylisothiocyanate derivatives are phenylthiohydantoins (PTH derivatives)

Dansyl Chloride

Step 3: Identify N- and C-terminal residues C-terminal analysis Enzymatic analysis (carboxypeptidase) Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys Carboxypeptidase B (hog pancreas) only works on Arg and Lys

Steps 4 and 5: Fragmentation of the chains Enzymatic fragmentation trypsin, chymotrypsin, clostripain, staphylococcal protease Chemical fragmentation cyanogen bromide

Polypeptide Cleavage Procedures

Enzymatic Fragmentation Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease C-side of Glu, Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer

Enzymatic Fragmentation The products of the reaction with trypsin are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the C-terminal end of the polypeptide.

Chemical Fragmentation with Cyanogen Br

Step 6: Reconstructing the sequence Use two or more fragmentation agents in separate fragmentation experiments Sequence all the peptides produced (usually by Edman degradation) Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain

Edman Degradation

Reconstructing a Sequence Compare cleavage by trypsin and staphylococcal protease on an unknown peptide: Trypsin cleavage of the unknown peptide gave: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease cleavage gave: F-S-G-I-T-P-K L-V-G-K-A-E

Reconstructing the Sequence of an Unknown Peptide Overlap of the two sets of fragments: L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E F-S-G-I-T-P-K Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K

Sequence analysis of catrocollastatin-C

Amino Acid Sequence Can Be Determined by Mass Spectrometry Mass spectrometry separates particles on the basis of mass-to-charge ratio Fragments of proteins can be generated in various ways MS can also separate these fragments

Amino Acid Sequence Can Be Determined by Mass Spectrometry

5.5 What is the Nature of Amino Acid Sequences? Sequences and composition reflect the function of the protein Membrane proteins have more hydrophobic residues, whereas fibrous proteins may have atypical sequences Homologous proteins from different organisms have homologous sequences e.g., cytochrome c is highly conserved Figure 5.16 illustrates the relative frequencies of amino acids in proteins.

5.5 What is the Nature of Amino Acid Sequences? Frequencies of amino acids in the proteins of the SWISS-PROT database.

Computer Programs Can Align Sequences and Discover Homology Between Proteins Alignment of the amino acid sequences of two protein homologs using gaps. Shown are parts of the amino acid sequences of the catalytic subunits from the major ATP- synthesizing enzyme (ATP synthase) in a representative archaea and a bacterium. These protein segments encompass the nucleotide-binding site of these enzymes. Identical residues in the two sequences are shown in red. Introduction of a three-residue-long gap in the archaeal sequence optimizes the alignment of the two sequences.

Blocks Substitution Matrix (BLOSUM) Methods for alignment and comparison of protein sequences depend upon some quantitative measure of how similar two sequences are. One way to measure similarity is to use a matrix that assigns scores for all possible substitutions of one amino acid for another. BLOSUM62 is the substitution matrix most often used with BLAST. BLOSUM62 assigns a probability score for each position in an alignment based on the frequency with which that substitution occurs in the consensus sequences of related proteins.

Blocks Substitution Matrix (BLOSUM) The BLOSUM62 substitution matrix provides scores for all possible exchanges of one amino acid with another.

Phylogeny of Cytochrome c The number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived This observation can be used to build phylogenetic trees of proteins This is the basis for studies of molecular evolution

Orthology in cytochrome c The sequence of cytochrome c from more than 40 different species reveals that 28 residues are invariant. When the sequences of a given protein from multiple organisms are homologous, they are said to be “orthologous”.

Related Proteins Show a Common Evolutionary Origin This phylogenetic tree depicts the evolutionary relationships among organisms as determined by the similarity of their cytochrome c sequences.

Related Proteins Show a Common Evolutionary Origin The sequence of cytochrome c is compared with an inferred ancestral sequence represented by the base of the tree on the previous slide. Uncertainties are denoted by question marks.

Related Proteins Show a Common Evolutionary Origin The amino acid sequences of the globin chains of human hemoglobin and myoglobin show a strong degree of homology.

Related Proteins Show a Common Evolutionary Origin Compare this structure with the structures of the β-chain of horse methemoglobin and that of sperm whale myoglobin.

Related Proteins Show a Common Evolutionary Origin Figure 5.21 Compare this structure with the structures of the α- chain of horse methemoglobin and that of sperm whale myoglobin.

Related Proteins Show a Common Evolutionary Origin This evolutionary tree is inferred from the homology between the amino acid sequences of the α–globin, β- globin, and myoglobin chains.

Apparently Different Proteins May Share a Common Ancestry Evolutionary relatedness can be inferred from sequence homology Consider lysozyme and human milk α-lactalbumin These proteins are identical at 48 positions (out of 129 in lysozyme and 123 in human milk α-lactalbumin Functions of these two are not related

Apparently Different Proteins May Share a Common Answer The tertiary structures of hen egg white lysozyme and human α- lactalbumin are very similar.

Similar structures, but different sequence and function The tertiary structure of hexokinase. Compare this structure with that of G-actin These two proteins have different sequences and different functions, but similar tertiary structures.

Related Proteins Share a Common Evolutionary Origin

5.7 Do Proteins Have Chemical Groups Other Than Amino Acids? Proteins may be "conjugated" with other chemical groups If the non-amino acid part of the protein is important to its function, it is called a prosthetic group. Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein, hemoprotein, flavoprotein.

5.7 Do Proteins Have Chemical Groups Other Than Amino Acids?

5.8 What Are the Many Biological Functions of Proteins? Many proteins are enzymes Regulatory proteins control metabolism and gene expression Many DNA-binding proteins are gene-regulatory proteins Transport proteins carry substances from one place to another Storage proteins serve as reservoirs of amino acids or other nutrients

5.8 What Are the Many Biological Functions of Proteins? Movement is accomplished by contractile and motile proteins Many proteins serve a structural role Proteins of signaling pathways include scaffold proteins (adapter proteins) Other proteins have protective and exploitive functions A few proteins have exotic functions

5.8 What Are the Many Biological Functions of Proteins?

Questions You should practice questions 1-8.