Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using carbon-13 NMR hydrogen/deuterium isotope shifts

Abstract

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers

“…The latter may not be physiologically relevant. Nevertheless, H͞D exchange rates have been measured for individual assigned amide protons of various membrane-associated peptides and proteins in detergent solutions (6)(7)(8)(9). Another NMR approach makes use of trapping techniques and subsequent transfer of the peptide from an aqueous membrane environment to isotropic media (10).…”

The orientation and dynamics of substance P in lipid environments

“…Measurement of amide proton exchange kinetics has also been used to probe the dynamics of peptides and proteins at various lipid surfaces (Henry et al, 1987;O'Neil & Sykes, 1988, 1989. In addition, shifts are seen in the pK,s of peptide titratable protons at SDS micelle surfaces (Fernandez & Fromherz, 1977;Woolley & Deber, 1987;O'Neil & Sykes, 1989;Beschiaschvili & Seelig, 1992).…”

Four‐bond deuterium isotope effects on the chemical shifts of amide nitrogens in proteins

“…three-bond, two-bond, and one-bond secondary chemical shifts. Higher-order (four-bond and five-bond) effects are usually too small to measure accurately: four-bond 13 C α ( 13 C β ) and 1 H N ( 1 H α ) deuterium isotope shifts of 6 ± 5(5 ± 7) ppb and À2 ± 1(À1 ± 1) ppb, respectively, have been reported in our recent studies. [27,29] Therefore, it came as a surprise when in a recent study, relatively large four-bond 15 N deuterium isotope effects ranging from 13 to 68 ppb and resulting from the 1 H-to-D substitution at the exchangeable amide position of the following residue have been reproducibly obtained in a pair of proteins.…”

“…[9][10][11][12][13][14] The development of multidimensional NMR [15,16] has had an impact on the number and accuracy of the measurements of a variety of deuterium isotope effects on chemical shifts. The effects of proton-to-deuterium ( 1 H-to-D) substitution on the chemical shifts of 13 C α , 13 C β , and methyl 13 C nuclei 1-to-3 bonds away [17][18][19][20][21][22][23][24][25][26] and, more recently, 1-to-3-bond effects on the chemical shifts of the backbone 15 N, 13 CO, 13 C α , 13 C β , 1 H N and 1 H α , [27][28][29] Tyr 13 C ζ [30] and Cys 13 C β nuclei [31] have been quantified. Hydrogenbonding effects on one-bond isotope shifts of backbone 15 N, [32,33] side-chain 15 N of Asn, and Gln [34] as well as 15 N and 1 H N nuclei in NH 3 groups of lysine side-chains [35] have been investigated.…”

“…[27,29] Therefore, it came as a surprise when in a recent study, relatively large four-bond 15 N deuterium isotope effects ranging from 13 to 68 ppb and resulting from the 1 H-to-D substitution at the exchangeable amide position of the following residue have been reproducibly obtained in a pair of proteins. [29] The relatively large magnitude of these effects (that is at least on a par with the magnitude of three-bond 13 C isotope shifts in proteins) warrants a further, more detailed investigation. 13 C four-bond deuterium isotope shifts in small conformationally constrained organic molecules have been analyzed previously, [3][4][5]7] and at least on one occasion, a very unusual four-bond deuterium isotope effect on proton chemical shifts (~150 ppb) resulting from a strong hydrogen bond between an enzyme and transition-state-analog inhibitor was observed.…”