Protein  Structure 

Proteins: Biological Function depends on conformation  

• Unique Primary Structure  = Unique 3D Structure

      (Covalent bonds)              (Noncovalent Interactions)

• Globular Proteins: water soluble, compact, hydrophobic interior / hydrophilic surface

      enzymes, receptors, carriers, hormones, etc. (dynamic agents)

• Fibrous Proteins: water insoluble, structural roles, extended structure

      collagen (tendons, bone), a-keratin (hair, nails), etc. (~static agents)

Four Levels of Description of (Native) Protein Structure

• Primary Structure: (~60-1000 amino acid residues)

         linear seq. of amino acid residues, covalent bonding including -SS-  

           (also called "covalent structure") definition

           (the primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms (including stereochemistry).  In general, polypeptides are unbranched polymers, so their primary structure can often be specified by the sequence of amino acids along their backbone. However, proteins can become cross-linked, most commonly by disulfide bonds, and the primary structure also requires specifying the cross-linking atoms, e.g., specifying the cysteines  involved in the protein's disulfide or other covalen bonds.)

• Secondary Structure:  definition

         local conformations of backbone, maintained by hydrogen bonds

• Tertiary Structure: 

         3D structure of a subunit (one polypeptide chain) in its native state

• Quaternary Structure: 

         Spatial arrangement of subunits in oligomeric proteins

• Denaturation: Partial to complete unfolding of native conformation

          Denatured Protein: Protein that has lost its native conformation

                      
                             This image is by Dr. George Helmkamp, Jr (UKMC).

Primary Structure

Protein Sequencing Strategies

   Older Methods

• reduce disulfide bonds (if any), beta-mercaptoethanol or dithiothreitol, separate chains

• N-terminal and C-terminal analysis

• cut protein into smaller pieces for sequencing with chemicals or proteases

       Trypsin (cleavage after Arg, Lys)

       Chymotrypsin (cleavage after aromatic residues, Phe, Trp, Tyr)

       Carboxypeptidase - cleaves C-terminal residue

       CNBr - chemical cleavage after internal Met residues

• separate and sequence fragments using Edman degradation
• find sequence from overlaps -- much like a giant jigsaw puzzle

   Newer Methods

• Mass spectrometry may be used for amino acid sequencing

• DNA sequencing (Genetic Code à Protein Sequence from ORF)

       Databases -  Nucleic acid sequences  /  Protein sequences

                    -  Saccharomyces: 12,068 kb, 5885 genes

                     - NCBI (Medline, GenBank, Entrez, Blast)

The f and j bond angles around the a-carbon can be plotted - Ramachandran Plots Most combinations are not possible due to steric hindrance the bulkier side groups (e.g. trp) have fewer allowed rotations glycine (no side group) will have fewer restraints  proline has a very restricted bond angle--tends to disrupt secondary structure

This image is  by Dr. George Helmkamp, Jr  (UKMC)

Peptide Bonds / Peptide Conformation

• Peptide bond: ~ double bond character; planar group, trans conformation (cis ? proline)

• Peptide conformations:  Phi / Psi ( f / y ) angles;  Ramachandran plots - “allowed” angles

         - Definition of Phi / Psi ( f / y ) torsion or dihedral angles. Note: The definitions below come from IUPAC - IUB rules for biochemical nomenclature (Biochemistry 9:3471 (1970)).  Some times you will find torsion angles defined differently but the values tabluated below will be identical.    

             f : torsion angle for N_i-C_ia    

  • f is considered (+) when the system is viewed down the central bond (N_i-C_ia) and the bond in front (C_i-1' - N_i) requires a rotation to the right (clockwise) in order to that it eclipse the bond to the rear atom (C_i').    

              y : torsion angle for  C_ia - C_i'

  • y is considered (+) when the system is viewed down the central bond (C_ia - C_i') and the bond in front (N_i-C_ia) requires a rotation to the right (clockwise) in order to that it eclipse the bond to the rear atom (N_i+1').

            f = y = 180^o (fully extended, planar conformation)

            f = -57^o ;   y = -47^o (right handed a helix);  f = +57^o; y = +47^o (left handed a helix)  

            f = -139^o ;   y = +135^o (antiparallel b sheet)

            f = -119^o ;   y = +113^o (parallel b sheet)

• Secondary Structure of Proteins (local folding / periodic structure of protein backbones)

          - a-helix : (Linus Pauling & Robert Corey - 1951;  Diff. a-keratin

                      pitch = 0.54 nm; rise 0.15 nm; 3.6 residues / turn

                      helix formers:  Met, Glu;   helix breakers: Pro, Gly

                      helical wheels / amphipathic helices:  (n, n + 4, n + 7, etc.)

  
              More views of the alpha helix. and a tutorial on the a-Helix

          

          - b-sheets : (Linus Pauling & Robert Corey - 1951)

                      antiparallel b-sheet  /  parallel b-sheet

         

                        Another view of the beta sheet and a tutorial on b-Sheet

   

          -Coils / Turns / Bends / Loops  

                     (~ 50% of the residues of an average protein; surface regions)

                   b-turns connect two antiparallel b-strands (type I, II) (x-P-G-x)

• recognizable combinations of a-helix and/or b-sheet occur in many proteins

          - bab unit     /    helix-loop-helix :

          - Hairpin loop:   ­ ¯         (antiparallel);     Cross-overs   ­  ­       ­  ­

          - Greek key motif :  ¯ ­ ¯ ­

          - b barrel

Tertiary Structure - 3D structure of polypeptide chain

• Forces Stablizing Tertiary Structure

          - Hydrogen bonding  (backbone and sidechain)

          - Hydrophobic interactions

          - "Ion pairs" - Electrostatic interactions

          - Disulfide bonds

• Tertiary Structure of Globular Proteins

          - hydrophobic AA generally on the inside

          - hydrophilic AA generally on the outside in contact with water

• Domains :  Conbination of motifs - 25 to 300 a.a. / function

          - b sandwich  /  b barrel  /  a/b barrel   /   helical bundle

• Functional units

          - Some Unusual Folding patterns of proteins are unique to their function

                  - left-handed parallel beta helix / horseshoe crab

          - Nucleotide binding domain babab / dinucl. binding domain = Rossmann Fold

          - Zn finger   /  Leucine zipper

Some Examples of Protein Structures (pink = helices; yellow = sheets)

Quaternary Structure - Arrangements of subunits in “oligomers”

•   a₄    ; a₁₂  ;    (ab)₂    ; (ab)₆

•  Many oligomeric proteins are allosteric (Mb vs. Hb - next unit) 

• 4-oxalocrotonate tautomerase (Monomer)	4-oxalocrotonate tautomerase (Dimer)	4-oxalocrotonate tautomerase (Hexamer)

More sites with protein structure tutorials:
           CMU Protein Architecture Site 
           Protein Architecture

Note: Some of the figures used in these class notes are copyrighted material from the text and are not to be used for any other purpose than to support this course.