Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Abstract

The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of sitespecific pK a s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine 13 C γ , 13 C δ2 chemical shifts and 1 H e -13 C e onebond scalar couplings. The methodology is first validated and then used to obtain pK a values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pK a values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.

Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes

“…Characterizing rare tautomeric and anionic bases in polynucleotides is a longstanding problem because such energetically unfavorable species typically exist in low abundance, for short periods of time, and involve movements of protons that are difficult to visualize at the atomic level. NMR relaxation dispersion (RD) techniques 20-22 are making it possible to characterize low-populated (populations of 0.1%-10%) transient (lifetimes of micro-to-milliseconds) states of nucleic acids 23-25 that are often referred to as ‘excited states’ (ES). Here, we use NMR RD to characterize transient WC-like dG•dT and rG•rU mispairs in DNA and RNA that are stabilized by rare tautomeric and anionic bases and obtain evidence that they play universal roles in misincorporation during replication and translation.…”

Tautomeric stabilities of 4-fluorohistidine shed new light on mechanistic experiments with labeled ribonuclease A

“…This may be one reason why synthetic incorporation of 4-fluorohistidine into RNase A was feasible and yielded an active enzyme: the fluorinated histidine analogue maintains the tautomeric preference of the enzyme. Meanwhile, the tautomeric form of His119 during proton transfer steps has not been definitively assigned, but recently developed methods for measuring histidine tautomers in transient protein conformations may be able to address this question[14]. …”

Limiting values of the one-bond C H spin-spin coupling constants of the imidazole ring of histidine at high-pH

“…In fact, only 106 13 C γ , versus 4,703 13 C δ2 , chemical shifts of the imidazole ring of histidine have been deposited in the Biological Magnetic Resonance data Bank (BMRB) [17]. Hence, problems in the determination of the chemical shifts for these nuclei, such as that for the ground state of His 40 in the protein Im7 [14], often prevent the use of this methodology; and ( iii ) the observed one bond C–H SSCC value at the high-pH limit is ambiguous, as will be discussed below.…”

“…The first problem pertains to the use of 1 J Cε1H SSCC to determine the protonation fraction of His, e.g., to detect sparsely populated, short-lived, protein states [14]. In detail, the low-pH limiting value for 1 J Cε1H SSCC appears to be quite well defined (221 ± 1.0 Hz [14]), for the 1 J Cε1H SSCC pH-dependence of four titrating His residues (His 6, His 13, His 26, His 87) of the PLCCγ SH2 protein domain [14,18].…”

“…In detail, the low-pH limiting value for 1 J Cε1H SSCC appears to be quite well defined (221 ± 1.0 Hz [14]), for the 1 J Cε1H SSCC pH-dependence of four titrating His residues (His 6, His 13, His 26, His 87) of the PLCCγ SH2 protein domain [14,18]. However, the observed high-pH limit for 1 J Cε1H SSCC differs among five His residues, of the PLCCγ SH2 protein domain, by up to ~6Hz [14], i.e., four titrating His residues converge to a high-pH limiting value of 207 ± 1.0 Hz while the remaining one (His 57), which is the only non-titrating His residue, shows an almost flat, pH-independent, value of 203 ± 1.0 Hz [14]. The existence of two possible high-pH limiting values for 1 J Cε1H SSCC, namely 207 ± 1.0 Hz or 203 ± 1.0 Hz [14], is a source of ambiguity.…”