The all-helical protein, DnaBN, exhibited EX1 type hydrogen exchange at pH 7.2. Approximately 45 protons were exchanged relatively rapidly, while an additional ∼50 protons exchanged more slowly. The rates of exchange for these slowly exchanging protons were the same, demonstrating that the slowest exchange events represent global unfolding. EX1 behavior is uncommon for native proteins. The protein was cyclized by joining the N- and C-termini through peptide linkers that were three, four, five or nine amino acids long. The corresponding "linear" proteins were extended by addition of the same amino acids to give proteins of identical amino acid composition as their cyclized versions but differing by the mass of a water molecule. All of the proteins unfolded approximately five times faster in 10 mM compared with 100 mM ammonium acetate. In all cases, the cyclized proteins showed slower rates of amide proton exchange related to global unfolding than their linear counterparts by a factor of approximately 7- to 12-fold. Interestingly, the rate of exchange for the slowly exchanging protons decreased for both the linear and cyclized proteins as linker length increased, and this correlated with predictions that the C-terminal helix of the protein would be extended by addition of these extra amino acids. This indicates that lengthening of this helix leads to a modest increase in stability of DnaBN. © 2010 Elsevier B.V.