1. Protein Folding: Theory and Experiment

• Proteins spontaneously fold into their native conformations under physiological conditions.
• A protein's primary structure dictates its three dimensional structure.
• Biological structures are self assembling.
• RNase A is a 124 residue single-chain protein.

AGILPV- aliphatics
FYW- aromatics
KRH - bases
DENQ - acids and corresponding amides
ST - alcohols - hydrophillic
CM - sulphurated

the colored Ribonuclease A for homo sapiens is

MALQRTHSLLLLLLLTLLGLGLVQPSYGQDG
MYQRFLRQHVHPEETGGSDRY
CNLMMQRRKMTLYH
CKRFNTFIHEDIWNIRSI
CSTTNIQ
CKNGKMN
CHEGVVKVTD
CRDTGSSRAPN
CRYRAIASTRRVVIA
CEGNPQVPVHFDG

If you are a cow the ribonuclease A coloring looks like:

KETAAAKFERQHMDSSTSAASSSNY
CNQMMKSRNLTKDR
CKPVNTFVHELADVQAV
CSQKNVA
CKNGQTN
CYQSYSTMSITD
CRETGSSKYPN
CAYKTTQANKHIIVA
CEGNPYVPVHFDASV

The lines have been broken at cysteine to emphasize the possibility of disulphide bonds.
On first inspection the peptide following the C's repeat in several instances.
Here is the primary and secondary structure:
 

Sequence and secondary structure

   1 KETAAAKFER QHMDSSTSAA SSSNYCNQMM KSRNLTKDRC KPVNTFVHES 
       THHHHHHH HHBTTTTSS   TTTHHHHHH HHGGGGTSS   SEEEEE ST 

  51 LADVQAVCSQ KNVACKNGQT NCYQSYSTMS ITDCRETGSS KYPNCAYKTT 
     HHHHHGGGGT EEE TTTT S  EEE SSEEE EEEEEETGGG BGGB  EEEE 

 101 QANKHIIVAC EGNPYVPVHF DASV 
     EEEEEEEEEE ETTTTEE EE  EE

A. Protein Renaturation

• Ribonuclease A spontaneously refolds when denatured,
  provided it is exposed to oxygen.
• This process can take several hours unless it is exposed to protein-disulfide isomerase (PDI). PDI speeds up the reaction to a few minutes.
  
• Postsynthetically Modified Proteins May Not Readily Renature
• Protein Folding is Directed Mainly by Internal Residues
• Helicies and Sheets Predominate Because They Fill Space Efficiently
• Sixty percent of proteins are alpha helices and beta sheets.
• A protein's internal residues direct its folding to the native confirmation.
• Protein folding is driven by hydrophobic forces.
• Native fold determines packing but packing doesn't determine native fold.
• There are a large number of ways in which a protein's internal residues can pack together efficiently.

B. Folding Pathways

• Time estimates do not include intermediate states.
  They do not include the likelihood of getting stuck in a metastable,
  but non-functional intermediate state.
• Bovine Pancreatic Trypsin Inhibitor (BPTI)
  Folds to Its Native Confirmation via an Ordered Pathway
• Consider 50,000 proteins in the body. There must be 50 K folding pathways as well. Sometimes there are multiple paths to a functional end state. These paths may have slow and fast conversion times.
• Each distinguishable disulfide-bonded species represents a subset of the conformations that the BPTI polypeptide can assume, so that by following the time course of the appearance of these various species, the approximate conformational path takn by the renaturing protein was deduced.
• Primary Structures Determine Protein Folding Pathways
• Protein folding occurs through an ordered pathway.
• Protein primary structures evolved to specify efficient folding pathways
  as well as stable native conformations.
• A protein's amino acid sequence dictates its native structure by specifying the series of steps comprising its folding pathway.

C. The Roles of Folding Accessory Proteins

• Most protein's renature in vitro.
• Protein Disulfide Isomerase (PDI) Facilitates
  Disulfide Interchange Reactions.
• Peptidyl Prolyl Cis-Trans Isomerases Facilitate
  the Formation of the Cis Peptide Bonds Preceding Pro Residues
• Molecular Chaperones Prevent Improper Folding and Aggregation.
• The Concept of Self-Assembly Must Take Accessory Proteins Into Account
   
  

D. Prediction of Protein Structures

• Chou and Fasman came up with a really clever way of looking at the frequency at which various amino acids tend to form or break alpha helices and beta sheet structures. It would be interesting to see if the same approach could be applied to longer aggregations of amino acids, to further refine the predictive capability. It would also be interesting to generate all the sequences of 2, 3, ... n peptides and compare the motifs that actually occur with those that could occur in a an exercise reminscent of the CTAG triplet table where 64 combinations occur, but only 20 + stop and start are unique.
• Reverse turns are characterized by a minimum in hydrophobicity along a peptide chain.
• Physical Basis of a Helix Propensity
  Brain Mathews answered this by cooking up 20 versions of a lysosyme from T4.
• Secondary structures are partially dictated by tertiary structures.

  2. Protein Dynamics

• Proteins are flexible and rapidly fluctuating molecules whose structural mobilities have functional significance.
• Proteins undergo breathing motions.
• There are three broad categories 1) atomic fluctuations, collective motions and triggered conformational changes.
• Proteins have mobile structures.
  A protein's native structure consists of a large collection of conformational substates that have similar stabilities.
• Protein core mobility is revealed by aromatic ring flipping
• Infrequent motions can be detected through hydrogen exchange
• The rate at which a particular proton undergoes hydrogen exchange is a reflection of the conformational mobility of its surroundings.
• Protein Folding Can Be Monitored by Pulsed Deuterium Exchange Methods