Though the changes in residue hydration are more prominent in PEG 5 kDa, the altered residues largely overlap between the two PEGylated proteins. We quantified the total number of hydrogen bonds between water and the backbone. Interestingly, the H-bond number of Gal3C, Gal3C-PEG 600 Da and Gal3C-PEG 5 kDa were all similar at 77.1, 76.5, and 76.4, respectively. This indicates that conjugation with a longer PEG does not dehydrate the protein surface and supports the notion that the differences observed in the behavior of Gal3C-PEG 5 kDa, compared to Gal3C-PEG 600 Da and unconjugated Gal3C in 2D IR and NMR experiments, are not attributable to replacement of water molecules. The H-bond lifetime, averaged among all the H-bonds formed between protein amide groups and the nearby water molecules (Figure ), quantifies effects of PEGylation on water dynamics around protein backbone. The H-bond autocorrelation exhibits a decay constant on the picosecond scale, which is associated with the process of H-bond formation and dissociation . Short PEG chain causes a ~10% slowdown in the lifetime while the long PEG chain leads to a ~30% slowdown, as compared to unconjugated Gal3C (Table ). In the dynamics analysis of 2D IR above, the frequency fluctuations in the amide I vibrational range can be interpreted as related to the rapid H-bond fluctuations between protein amide groups and nearby water molecules . The computed H-bond lifetime confirms our measurements of perturbed H-bond fluctuations upon PEGylation, as evidenced by the extended H-bond lifetimes computed from MD simulations. Moreover, the time scales of computed H-bond lifetimes are comparable to the relaxation time extracted from CLS analysis of 2D IR, where the slowdown in H-bond lifetime qualitatively agrees with the slower dynamics observed in the 2D IR experiments for Gal3C-PEG 5 kDa (Figure ).