ALBERT

Notes: Two dimensional nmr spectra [correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA), nuclear Overhauser effect spectroscopy (NOESY)] have been observed for cross-linked lysozyme, a chemically modified lysozyme derivative with an extra ester cross-link between residues E35 and W108. Eight shifted cross-peaks were found in the fingerprint region of COSY spectra. By searching COSY, HOHAHA and NOESY spectra, they have been assigned to A32, E35, S36, I58, A107, W108, V109, and A110. The NOE connectivities (dNN and dαN) found for the cross-linked lysozyme are quite similar to those for the intact lysozyme.Exchange behavior of amide hydrogens has been studied for both intact and cross-linked lysozymes by observing the fingerprint region of COSY spectra. Hydrogen exchange reactions were carried out at pH 7.0 and at several temperatures. There exist 41 amide hydrogens whose exchange reactions are detectable under this experimetal conditon. Not only exchange rates but also their activation enthalpies were determined for individual amide hydrogens. They are classified into two groups, which are called categories III and IV. Category III hydrogens are distributed in relatively flexible peripheral parts of protein, and category IV hydrogens are deeply buried in the core region of protein. Category III hydrogens are exchanged through localized unfolding around their sites with a low activation enthalpy ranging from 10 to 25 kcal/mol. The formation of an extra cross-link affects neither the exchange rate nor the activation enthalpy of category III hydrogens. However, amide hydrogens of residues 34-39 in the vicinity of the hinge are exceptions. They are easily exchanged in the intact lysozyme but their exchange rates are drastically retarded by cross-linking. In the intact lysozyme, structural fluctuations mediating the exchange of category IV hydrogens are highly cooperative with a large activation enthalpy. These large-scale structural fluctuations are the global unfolding of the overall structure and also concerted motions within a domain. Especially near 38°C, it was found that the dominant fluctuation occurring in the α-domain is different from that in the β-domain. However, these concerted motions are strongly quenched by the formation of the cross-link because of the cooperativity of such a large-scale fluctuation. The stabilization of a localized area of protein by cross-linking results in the great suppression of large-scale and concerted motions. The exchange rates of category IV hydrogens are extremely retarded in the cross-linked lysozyme, so that they are exchanged through the so-called penetration mechanism characterized by a low activation enthalpy. These expeimental results are discussed with regard to the contribution of cross-linking to the stabilization of the folded structure of protein. © 1997 John Wiley & Sons, Inc.