The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo-and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo-and polypeptides. The application of this approach to protein studies is discussed.

The hydrogen exchange behavior of native cytochrome c in low concentrations of de-naturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.Under native conditions, a small fraction of any population of protein molecules occupies each possible higher energy, partially unfolded state, including even the fully unfolded state, as described by the Boltzmann distribution. The study of these partially unfolded forms (intermediates) may illuminate the fundamental cooperative nature of protein structure and define the unfolding and refolding pathways of a protein even though the intermediates are normally invisible to measurement. The energy levels and therefore the occupation of these conformationally excited states can be manipulated by denaturants and temperature. Hydrogen exchange experiments can then determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to the perturbant. From this we can infer, respectively, the structure, the free energy, and the surface exposure of each protein form.Results for cytochrome c reveal a small sequence of distinct partially unfolded forms with progressively increasing free energy and degree of unfolding. These appear to represent the major intermediates in the unfolding and refolding pathways of cytochrome c. Hydrogen exchange theoryExchangeable amide hydrogens (NH) that are involved in hydrogen-bonded structure can exchange with solvent hydrogens only when they are transiently exposed to solvent in some kind of closed to open reaction (1-3), as indicated in Eq. 1.(1)In the almost universally observed limiting case, referred to as EX2 (for bimolecular exchange) (1), the structural opening reaction enters the rate expression as a pre-equilibrium step. The exchange rate of any hydrogen, k ex , is then determined by its chemical exchange rate in the open form, k ch , multiplied by the equilibrium opening constant, K op (Eq. 2).

Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Key to kinetochore assembly is the specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A/H2B. We further find that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through nucleosome-docking-facilitated hydrophobic interactions.