The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47 â Glu has been investigated by nuclear magnetic resonance (NMR) spectroscopy at 600 MHz. The ¹H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in the authors's previous paper were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds. As found previously, the orientation of the exposed finger of antiparallel β-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core. The Lys-47 â Glu mutation has only a small, but clearly discernible, effect on the structure which can be attributed to the larger space requirement for the longer Lys side chain relative to that of the shorter Glu side chain.