112 Hydrogen exchange and protein folding Jane Clarke* and Laura S Itzhakit Amide hydrogen-deuterium exchange is a sensitive probe of the structure, stability and dynamics of proteins. The significant increase in the number of small, model proteins that have been studied has allowed a better understanding of the structural fluctuations that lead to hydrogen exchange. Recent technical advances enable the methodology to be applied to the study of protein-protein interactions in much larger, more complex systems. the 'open', exchange competent form. K op is the rate constant for the opening step, k cl is the rate constant for the closing or reproduction step. kine is the rate constant for the chemical exchange reaction, which will depend on the protein primary sequence, pH and temperature. Under folding conditions, kcl»K op , so that the observed rate constant for exchange, k ex ' can be defined as In the other limiting case, EX1, the chemical exchange step is much faster than the rate constant for reprotection and so the opening step becomes rate-limiting: (3) (5) (2) (4) kop·kine kcl + kine k ex = k op k ex where K op is the equilibrium constant for the opening reaction. This is known as the EX2 limit. The apparent free energy of exchange, can be calculated assuming kine, which depends on pH, temperature and position in the protein, can be accurately inferred from peptide studies [5,6]. The two-process model In the generally accepted two-process model, originally proposed by Woodward and co-workers [3,4], protected amide protons can exchange through relatively rare global unfolding events, or through fluctuations of the native state. The nature of the native state fluctuations are still unresolved, but most investigators assume' the two-step, model when analysing both global and native-state exchange kinetics. EX1 and EX2 kinetics From equation 2, there are two limiting conditions for exchange. If the open species equilibrates rapidly with C, so that the closing step, k e ], is faster than the exchange step kint' the the chemical exchange is the rate-limiting step, and k ex reduces to where R is the gas constant and T is the temperature in Kelvin. Exchange under EX2 conditions, therefore, can be used to determine the apparent free energy of the underlying structural opening reaction. (1) Current Opinion in Structural Biology 1998, 8: 112-118 http://biomednet.com/elecrefl0959440X00800112 © Current Biology Ltd ISSN 0959-440X Abbreviations BPTI bovine pancreatic trypsin inhibitor CI2 chymotrypsin inhibitor 2 DHFR dihydrofolate reductase GdmCI guanidinium chloride HX hydrogen exchange OMTKY3 ovomucoid third domain Introduction Hydrogen exchange (HX) is a powerful technique that has been used for many years to study the structure, stability, folding, dynamics and binding of proteins. The pioneering work on HX focused on a small number of proteins. In the past few years, this has been extended to include a significant number of small, model proteins, many of which lack the disulphide cross-links or prosthetic groups that can complicate the analysis. The combination of HX and other folding studies of these systems has improved our understanding of the conformational dynamics of proteins at equilibrium. In this review, we concentrate on HX at equilibrium. We do not have room to discuss kinetic studies of protein folding, nor HX in denatured states. Addresses ·tCentre for Protein Engineering, MRC Unit for Protein Function and Design, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK 'Present address: University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK; e-mail: jc162@cam.ac.uk te-mail: Isi@mrc-Imb.cam.ac.uk Theory of the hydrogen exchange mechanism Hydrogen exchange mechanism The commonly accepted model is the two-step model of [1], refined by Hvidt and Nielsen [2], and described as follows Hand 0 denote protonated and deuterated forms. C denotes the 'closed', exchange incompetent form, and 0 EX1 and EX2 exchange mechanisms can be distinguished from the pH dependence of exchange, from the decay of