Biochemistry. Author manuscript; available in PMC 2012 March 8.
Published in final edited form as: Biochemistry. 2011 March 8; 50(9): 1429–1431. doi:10.1021/bi102057m.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Large Favorable Enthalpy Changes Drive Specific RNA Recognition by RNA Recognition Motif Proteins†
Krystle J.McLaughlin, Jermaine L. Jenkins, and Clara L. Kielkopf* Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
The RNA recognition motif (RRM) is a prevalent class of RNA binding domain. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively few. Here, we use isothermaltitration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF65, (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (−30 to −60 kcal mol−1) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearlyphysiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs. The RNA recognition motif (RRM) is an abundant domain among proteins with central roles in post-transcriptional gene regulation (1) (Figure 1). Multiple RRMs often occur per polypeptide. For example, tandem RRMs of the splicing factors U2AF65, TIA-1 and SXL recognizeU-rich pre-mRNA sites (2,3). The poly(A) binding protein (PAB) enhances translation following RRM-mediated recognition of the mRNA tail (4). Beyond the wellestablished role of canonical RRMs to bind RNA, RRM variants such as the U2AF Homology Motif (UHM) are dedicated to protein-protein interactions (5). This breadth of functions illustrates the importance of elucidating the structural andthermodynamic forces responsible for RRM interactions. Despite extensive structural and functional investigations of RRMs, full thermodynamic characterizations of the enthalpy and entropy changes during RRM/RNA binding are scarce. A thorough characterization of RNA binding by an RRM has been completed for the U1A splicing factor (6). However, this rare example focuses on a single RRM binding a singlestranded(ss)RNA site within a stem loop, which differs from the prevalent systems of multiple RRMs recognizing unstructured ssRNAs. Enthalpy and entropy changes of ssRNA binding have been determined for a number of unrelated domains, including Hfq, GLD-1, trp attenuation protein (TRAP), tristetraprolin, and T4 translational regulatory protein (7– 11). Given the diversity of these domain classes, it isunsurprising that no common thermodynamic themes of ssRNA binding have emerged to date. In contrast, protein/protein, protein/ligand, and protein/DNA interactions have been investigated in depth. Previously, we found a large favorable enthalpy change (ΔH) and a large unfavorable entropy change (−TΔS) for poly(U) recognition by the tandem RRMs of U2AF65 (ΔH −70
†This work was supported by theNational Institutes of Health (Grant GM070503 to C.L.K.).
To whom correspondence concerning the manuscript should be addressed: C.L.K.: firstname.lastname@example.org; phone: 585-273-4799; fax: 585-275-6007. SUPPORTING INFORMATION AVAILABLE Table S1, Figures S1–S5, and Supplementary Methods. This material is available free of charge via the Internet at http://pubs.acs.org
McLaughlin et al.Page 2
kcal mol−1, −TΔS 61 kcal mol−1) (12). Notably, these enthalpy and entropy changes were similar in magnitude to those observed for the single other example of RRM/RNA binding (U1A RRM/RNA stem loop, ΔH −68 kcal mol−1, −TΔS 58 kcal mol−1) (6). To explore the generality of these observations for other RRM-containing proteins, we used isothermal titration calorimetry (ITC) to determine...