Nomenclature - classes of proteins
        Historically based on SOLUBILITY of PROTEINS...    Two classes - SIMPLE & COMPLEX
  
        SIMPLE PROTEINS: on hydrolysis include only amino acids:
            1. Albumins - soluble in water (distilled),  globular,  most enzymes
            2. Globulins - soluble in dilute aqueous solutions; insoluble in pure distilled water
            3. Prolamins - insoluble in water; soluble in 50% to 90% simple alcohols
            4. Glutelins - insoluble in most solvents; soluble in dilute acids/bases
            5. Protamines - not based upon solubility; small MW proteins with 80% Arginine & no Cysteine 
            6. Histones - unique/structural: complexed w DNA,  high # basic aa's - 90% Arg, Lys, or His
            7. Scleroproteins - insoluble in most solvents
                                     fibrous structure - architectural proteins of cartilage & connective tissue
                      Collagen = high Glycine, Proline, & no Cysteine when boiled makes gelatin
                      Keratins - proteins of skin & hair high basic aa's (Arg, His, Lys), but w Cys
 
 

 

 

 

 

    

 

 

 

 

 

 
  Structure of Proteins 
  
  the Variety of Protein Structures may be INFINITE...
         average protein has 300-400 amino acid's  &  has a MW of 30kD to 45kD
          a PROTEIN of 300 amino acids made with 20 different kinds 
          of amino acids can have 20³⁰⁰ different linear arrays of aa's  [10³⁹⁰ different proteins]

1st protein sequenced was Beef  Insulin           by Fred Sanger - 1958 Nobel Prize winner                2 polypeptides [21/30 aa's]       Humulin   &  ADA           to date about 100,000 human proteins are suspected           only about 10,000 structures have been identified...                                           E. coli make about 3,000 proteins.

     

 

 

 

               4 levels of protein structure are recognized
 

	primary      - linear sequence of aa's
	secondary  - regular, recurring orientation of aa                           in a peptide chain due to H-bond
	tertiary      - complete 3-D shape of a peptide                           due to weak electrostatic forces
	quaternary - spatial relationships between different                           polypeptides or subunits

 

 

 

 

 

 

 

 

Primary sequence…

Linear sequence of amino acids in a polypeptide                                               repeated peptide bonds form the back bone of the polypeptide chain                   R side groups project outward on alternate sides

Chain... one end of polypeptide chain has a free (unlinked) amine group:  N-terminus                      other end has a free (unlinked) carboxyl group: C-terminus                                  N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C

Size… a protein's size is specified by its mass (MW in Daltons = 1 amu)                     average MW of a single amino acid ≈ 113 Da                                thus if a protein is determined to have a mass of 5,763 Da  ≈  51 amino acids                    average yeast protein  =  52,728 Da  [52.7 kDa] with about 466 amino acids

Protein Primary Sequence today is determined by reading the GENOME Sequence

Protein function is derived from the 3D structure (conformation) specified by                    the primary amino acid sequence and the local environs interactions.   [lysozyme*]

 

 

 

 

 

some consequences... of Primary Sequence…

Polymorphism... proteins may vary in primary sequence but still                         exhibit the same catalytic activity.    ex: peroxidase... H2O2  -->  2 H2O + O2                                                      inter-specific:   between species  [have diff.  aa sequences]                                                intra-specific:   within a species   [ liver vs. kidney ]

Invariants... don't vary significantly in aa sequence                                                  examples:   ubiquitin (proteosomes)   &    histones (chromosomes)

Site Specificity… unique sequences determine intra-cellular location & function                                       signal sequences of protein targeting, prosthetic binding sites, etc…

Families of Proteins: different structure but with related functions                                evolved from a single ancestral protein, up to 30%+ commonality of sequence...                            serine proteases (trypsin,  chymotrypsin,  elastase) all have SER at active site.

Homologous Proteins… similar characteristics: structurally similar; may  perform the same                   cellular function, often in different species & related by common evolutionary history;                   ex: cytochrome-C: in duck & chickens = 2 variants  &  in yeast & horses = 48 variants

Mutation - change in primary amino acid sequence = defective protein - SICKLE CELL

 

 

 

 

 
Secondary structure - well defined periodic structure: makes up 60% of a protein's structure
   
   Alpha helix*   described by Linus Pauling 1954 Nobel    using  X-ray*diffraction technique   

rigid rodlike cylinder around long axis core
R-groups radiate outward
3.6 aa per 360o turn
single repeat turn of helix (360o) = 0.54 nm
forms right handed helix - (counterclockwise)
helix formed from H-bond interactions
H of N  (of one aa)   &   -C=O  (of 4th aa)
¼ of aa's in globular proteins occur in alpha helix
flexible - wool is stretchable (breaks H-bonds)
mcb fig 3.4*	click*

 

 

 

 

 

  

 

 

 

 

     EF hand...  two short helices connected by a loop; a Ca⁺² ion binder region of hydrophilic
                     residues present in over 100 Ca⁺² binding proteins... aka                       fig 3.9b* 
          helix loop helix... commonly bind gene transcription factors to DNA
   
     zinc finger...  1 a and 2 β  strands with antiparallel orientations.
                         forms fingers bound by Zn ion that often link to DNA (RNA)            fig 3.9c* 
   
     coiled coil...   a helicies, where the hydrophobic amino acids in one helix wind together
                           forming a coil with others; also called leucine zippers due to high
                           [leu]: also common to transcription factors.                                    fig 3.9a*
   
                    Prints:  a protein fingerprint database of conserved protein motifs

 

 

 

 

 

 

 
 Tertiary level
        level most responsible for 3-D orientation of proteins in space 
               is the thermodynamically most stable conformation of a protein... and is due to
                         – weak non-covalent interactions  [ figure* ]  
                         - hydrophobic interior & hydrophilic exterior favors globular shapes
                         - via H-bonds (ecb fig 4.31*)
                         - & S-S bridges  fig 16.19*          [ecb fig 4.29]
               results in Protein Folding into specific 3D shapes & unique binding sites   ecb fig 4.9  
        some examples of 3D structure in proteins: 
                 Lysozyme        MW 14,600 enzyme; egg white & human tears  pdb-lysozyme
                                                                124 aa's with  4 S-S;  that hydrolyses polysaccharies
                                                   in bacterial cell walls = bactericidal agent   catalog   
                 Myoglobin         MW 16,700 - animal muscle protein - stores O₂  pic
                 Cytochrome -C  MW 12,400 -  heme binding single   pic
                                                     polypeptide of 100 aa's in ETS of mitochondria 
           Ribonuclease   MW 13,700 enzyme of 124 aa w 4 S-S  pic
  
 

 

 

  DOMAINS - distinct modules or structural elements of the tertiary level of protein structure...
                      compact folded regions in a polypeptide of 100-150 amino acids, often self-forming,
                      self-stabilizing, that often fold independently.           ecb 5.12* & ecb 5.13*
  3 classes of domains:
     functional domain - region with particular activity characteristic of a protein CATALYSIS:
                ex: tyrosine kinase* activity domain of human insulin that add P~ to other molecules. pic
     structural domain - region of 40+ aa's in a stable 2nd or 3rd-ary conformation, often repeatable.
                         ex: 1. hemagglutinin: -  a surface protein on influenza viruses, that is made of 3
           mcb 3.10a*      quaternary identical subunits composed of 2 polypeptides (HA₁ & HA₂);
                                  each HA peptide has two domains... a globular domain and a fibrous domain  
                              2. EGF (Epidermal Growth Factor) domain - a small soluble peptide hormone
           mcb 3.11*         that binds to embryonic cells in skin/connective tissue & promotes cell division.
                                  EGF is generated by proteolytic hydrolysis as a domain from several other
                                  proteins, all of which have an EGF domain as a structural part.
     topological domain - distinctive spatial relationships to rest of a protein;
                                  ex: membrane proteins with extrinsic cytoplasmic domain   (CD4 protein pic)
                                 and intrinsic membrane spanning domain.
     

 

 

 

 

 

 

 

 

 
  PROTEIN FAMILIES -  proteins with a common evolutionary ancestry...
              some proteins have many identical or chemically similar amino acids in identical
              sequence positions; each may contain domains that closely resembles that of other proteins.
  
              Proteins with common ancestors are known as homologs & belong to a "family".
         protein family: proteins with evolutionary relationships
                                (>30% aa sequence homology or common descent)
                     ex: 1.  serine proteases  ecb fig 4.21* - proteolytic enzymes with nearly identical
                                                            amino acid sequences all with a SER at the active site 
         protein superfamily: proteins with a probable common evolutionary origin that generally
                                                            contain one or more common motifs or domains
              family relations often best displayed by taxonomic cladistics (tree diagrams)
                       globins - gene slowly diverged into animal and plant lineages                  mcb 3.13a*
                                                        myoglobin - monomeric oxygen binder of muscle
                                                        hemoglobin - tetrameric oxygen binder of blood   mcb 3.13b*
  
                     Today, computer modeling is used to predict function of yet unisolated proteins
               by comparing known sequence homologies     sequence analysis = 2ndary structure  end 8
 

 

 

 

 

 
 QUARTERNARY structure: multiple polypeptides each with a 3-D conformations = final shape
                           ex:   hemoglobin (pic*),     RNA polymerase,     ASP-trans-carbamylase

Some Common Quarternary Level Protein Shapes...
1. dimers -   self recognizing symmetrical regions - bind together @ identical binding sites
                  [ Catabolic Activator Protein* ]   homodimers - 2 identical subunits
                                                                    heterodimers - non-identical subunits (as in PDH)
2. filaments  - polymers of protein subunits each bound together in an identical way
                             forming a ring or helix      see ecb 4.24*
3. colied-coil - 2 parallel helicies forming a stiff filament, linked via  
                             a stripe of hydrophobic aa's.     gk 4.16   [ keratin- ecb 4.16* ]
4. tetramers -  4 identical subunits... ex: neuraminidase - ecb 4.22* and hemoglobin*
 

    Multi-Enzymes Complexes : hemagglutinin A - a trimer of 3 identical polypeptide units  fig 3.10b*
                                             pyruvate dehydrogenase    picture*   &    pic
                                       ATP-synthase                   figure*  

  

 

 

 

 

 

  
 Multimeric proteins can have very complex...  Quartnerary Structure...

                 and  form very large Macromolecular Assemblies... 
                 ( > 1m Da in mass ),    30-300 nm in size,    &    10-100 individual peptides

                 examples include: 
                                            viral capsids, some cytoskeletal complexes, molecular machines,
                                            and mRNA transcription complex (some 60 proteins - fig 3.12*)

                 examples of such Molecular Machines can be seen in mcb/5e - table 3.1*
                                                                  we will look at some of these in greater detail later...

                 summary figure of protein structure - mcb 3.2*
 
                         

                                                                                 

 

 

 

 

 

 

 

 

 

 

 

 

 

protein shape & conformation...

NATIVE Protein CONFORMATION is the…                       3-D SPATIAL ORIENTATION that is MOST thermodynamically STABLE                                       and has the lowest free energy expenditure (forms spontaneously)       3 most common conformations HELIX - a spiral staircase-like shape                                            •              FIBER - elongated bound monomers                                           GLOBULAR - roughly a sphere

the Native Conformation of most enzyme proteins is GLOBULAR:                                       an interior pocket of hydrophobic amino acids                                       an exterior surface of hydrophilic amino acids                                                                   - maximizes the number H-bonds that form   fig 5.5*

the PHYSICAL forces include mostly weak electrostatic bonds*:                    non-covalent bonds, H-bonds,  hydrophobic  &  hydrophilic                    interactions, & covalent bonds (as peptide bonds & disulfide bonds)

results in a great variety of protein shapes & sizes - ecb 4.9 pg 127*

 

 

 

 

 
 How does 3D protein folding come about?             "FUNCTION follows FORM"
  
 peptide bond is PLANAR (partial double bond character) as are all the atoms bonded to it
 all occur is same plane* & thus there is no free rotation = restricts protein conformations
    
  µ the native folded conformation is most stable, i.e., in lowest free energy state, often
       dictated by R-group properties (size, hydrophobicity) hydrophilicity, ionic strength
   
       folding involves: changes in 3D conformations:
                - by orderly steps in a sequential way, each step facilitating the next -
                - first 2⁰ structure (a & β), then structural motifs & assembly of complex domains,
                         followed by 3⁰ level forces and/or 4⁰ shapes.              fig 3.15*
  
       Unless protected during folding, proteins would interact with all the other molecules in a cell.
  
 Cells makes 2 sets of proteins that facilitate folding: 
        Molecular Chaperones - which bind and stabilize newly made unfolded proteins preventing these
                         proteins from self aggregating and/or being denatured before folding.
        Chaperonins - which makeup a small folding chamber into which unfolded proteins are moved
                         to provide a proper environment favoring native folding of a protein.
 

 

 

               
     
MOLECULAR CHAPERONES - are families of proteins to help "properly fold" a new protein...
          multiple chaperones bind to newly made proteins and include:
               Hsp70 (of cytosol & mitoplasm);     BiP (of the E.R.);  &     DnaK (of bacteria).
     
   1st discovered via heat shock treatment [via temperature elevation 25^o --> 32^oC]
        by Ferruccio Ritossa (1962 - Italy) in heat shocked fruit flies =  Chromosome puffs
   all cells make heat shock proteins (HSPs); but mutant bacteria didn't make Hsp's
                                                                         nor did they assemble normal proteins.
  
   HSP's are ubiquitous to all cells - produced in response to stress (heat, infection, etc...)
              and they act as "Chaperones" for other proteins by:
                           1. inhibiting undesirable interactions with other proteins
                           2. promoting desirable interactions
                                          help form stable bonds between protein partners
                                          in establishing proper conformation & preventing aggregations.
      

 

 

 

 

 

 

 
   

  Classes of Heat Shock Proteins:    HSP -40, -60, -70, -90  &  -100.
                     HSP are named according to the molecular weights (70 = kilodaltons)

  HSP-40 binds new protein amino acid chains & carries it to Hsp-70

   Hsp-70 grabs proteins by an open cleft when ATP is bound to it
                 OPEN conformation has hydrophobic pocket for new unfolded protein... 
                 in its ADP conform closes around protein and aids native folding...  mcb6e.3.16*

   HSP-90 receives partially folded proteins from Hsp-70's and other chaperones...
                    helps join polypeptides into larger quaternary proteins forming
                    multi-subunit proteins, such as cellular receptors.                 figure*

             protein folding animations*^{view @ home}                      protein folding video*

 

 

 

 

 

 

 

 
 HSP-60  also know as CHAPERONINS  or foldase -
                      is a small folding CHAMBER of HSP's into which unfolded proteins are
                      moved to provide a proper environment favoring native folding...       figure*
  
     a Molecular Machine: made of chaperone proteins  hsp70's & 60's form barrel shaped structure
                made of 14 polypeptides (from GroEL gene) in 2 donut rings with a cap (from GroES gene)
                that opens an inner chamber, where a cell's new protein enters & is folded.
        barrel chamber has 2 conformations: tight & relaxed; 
        new peptide is inserted into cavity of GroEL chamber & conformational changes favor native
        protein folding; ATP hydrolysis = relaxed state & release of native 3D-protein  mcb6e-fig 3.17*
             fig A  &  fig B  [A.L. Horwich: PNAS 96:11-37, 1999] 

  HSP 100  - also known as unfoldase; also has a multi-subunit ring structure;
                     along with HPS-70 disassembles degraded proteins.

    

 

 

 

 

 

 

 

Misfolded Proteins  &  Disease
  PRION:     a defective protein agent (PrP^sc) due to mis-coded gene (PRNP^c)
                           native prion protein is PrP^c & resides on nerve cell surfaces...
                           defective protein PrP^sc accumulates forming aggregates that lead to CJD & SE's
   CJD:     Creutzfeld-Jacob disease, genetic based or acquired - (by eating "mad cow" tissue)
                                fatal neurological disease due to presence of misfolded PRP^c protein.
               Spongiform Encephalopathy (SE) - vacuolation (holes) in brain nerve tissue  

Both PRION proteins can have identical aa sequence,     but may fold differently        [are conformers = proteins differ only in conformation] A.  normal (PrPc) protein...          mostly α-helix foldings - remains soluble  B. abnormal PrPsc protein...          45% β-sheet - insoluble & protease insensitive          produces cell surface aggregates that kill cells              mechanism of action chart           McGraw-Hill Online Learning - Raven et al 7th edition

 

 

 

  

  PROTEIN DEGRADATION (Digestion/Turnover)..  getting rid of misfolded proteins

    cells often contain specialized mechanisms or pathways to digest cell proteins...
           1.  to rapidly turnover proteins with short half-lives
           2.  to recognize & eliminate damaged or misfolded proteins that can lead to diseases
                           as Huntington's, Alzeheimer's, and Creutzfeldt-Jacob disease. 
 
    a. many proteins are degraded in cytosol using PROTEASES to cut (hydrolyze) peptide bonds

    b. some proteins are degraded in the LYSOSOMES via phagocytosis,

    c. but most proteins are degraded by large complexes of proteolytic enzymes in structures
             known as PROTEASOMES in process known as Ubiquitin-mediated Proteolysis (UMP).

                    short half-life proteins hold a signal sequence targeting proteins for UMP
                    and misfolded proteins seem to be recognized for degradation by the UMP.

                                                         protein digestion by proteasomes*

 

 

 

 

 

Discovered by Alfred Goldberg & Martin Rechsteiner