The ras binding domain (RBD) of the Ser/Thr kinase c-Raf/Raf-1 spans 78 residues and adopts a structure characteristic of the beta-grasp ubiquitin-like topology. Recently, the primary sequence of Raf RBD has been nearly exhaustively mutated experimentally by insertion of stretches of degenerate codons, which revealed sequence conservation and hydrophobic core organization similar to that found in an alignment of beta-grasp ubiquitin-like proteins. These results now allow us to examine the relationship between sequence conservation and the folding process, particularly viewed through the analysis of transition state (TS) structure. Specifically, we present herein a protein engineering study combining classic truncation (Ala/Gly) and atypical mutants to predict folding TS ensemble properties. Based on classical Phi-value analysis, Raf RBD TS structure is particularly polarized around the N-terminal beta-hairpin. However, all residues constituting the inner layer of the hydrophobic core are involved in TS stabilization, although they are clearly found in a less native-like environment. The TS structure can also be probed by a direct measure of its destabilization upon mutation, DeltaDeltaG(U-++). Viewed through this analysis, Raf RBD TS is a more diffuse structure, in which all residues of the hydrophobic core including beta-strands 1, 2, 3 and 5 and the major alpha-helix play similar roles in TS stabilization. In addition, Phi-values and DeltaDeltaG(U-++) reveal striking similarities in the TS of Raf RBD and ubiquitin, a structural analogue displaying insignificant sequence identity (<12%). However, ubiquitin TS appears more denatured-like and polarized around the N-terminal beta-hairpin. We suggest that analysis of Phi-values should also consider the direct impact of mutations on differences in free energy between the unfolded and TS (DeltaDeltaG(U-++)) to ensure that the description of TS properties is accurate. Finally, the impact of these findings on the modeling of protein folding is discussed.